Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes

ABSTRACT

A complex includes: (1) a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and (2) a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of the probe and the target is purified or synthetic; and (b) the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins. A method for assaying a target includes detecting formation of the complex.

BACKGROUND OF THE INVENTION

1. Field of Invention

The invention relates to nucleobase binding in complexes, such asduplexes, triplexes and quadruplexes, and more particularly to methodswherein such complexes are formed by specific binding betweensingle-stranded or double-stranded nucleobase-containing probes andsingle-stranded or double-stranded nucleobase-containing targetsequences.

2. Description of Related Art

The Watson-Crick model of nucleic acids has been the accepted standardin molecular biology for nearly fifty years. As recounted by JamesWatson in his book entitled “A Personal Account of the Discovery of theStructure of DNA,” (1968), the Watson-Crick model, which won Watson andCrick the Nobel Prize, arose from the ashes of their abandoned theorythat bases bind to like bases on opposing strands (Watson at p. 125).Watson described how he abandoned his “briefly considered like-with-likepairing” model when he realized the advantages of a model based on A:Tand G:C binding. Id.

Although antiparallel nucleic acid duplexes first suggested by Watsonand Crick are the most widely studied type of multiple-strand nucleicacid structures, it has been discovered that nucleic acids also formtriplex structures and quadruplex structures under certain conditions.

Until recently, binding among three nucleic acid strands to form atriplex was widely believed to be confined to very limited species ofnucleic acids (e.g., polypurine or polypyrimidine sequences). See, e.g.,Floris et al., “Effect of cations on purine-purine-pyrimidine triplehelix formation in mixed-valence salt solutions,” 260 Eur. J. Biochem.801-809 (1999). Moreover, canonical triplex binding or hybridization wasthought to be based on Hoogsteen binding between limited varieties ofadjacent nucleobases, rather than Watson-Crick base pairing. See, e.g.,Floris et al. and U.S. Pat. No. 5,874,555 to Dervan et al. However, theinventors have recently disclosed in several patent applications thatspecifically bound mixed base sequence triplex nucleic acids based onWatson-Crick base pairing can be created and used as the basis for ahighly accurate and sensitive assay for specific binding. See U.S.patent applications Ser. Nos. 09/613,263 and 09/468,679, respectivelyfiled Jul. 10, 2000 and Dec. 21, 1999.

Zhurkin et al., 239 J. Mol. Biol. 181 (1994) discloses the possibilityof parallel DNA triplexes; however, these triplexes are said to becreated by the third strand binding in the major groove of the duplex inthe presence of recombination proteins, such as RecA protein.

As has been the case with triplex nucleic acids, the conventional wisdomregarding quadruplex nucleic acids has been that such peculiarstructures only exist under relatively extreme conditions for a narrowclass of nucleic acids. In particular, Sen et al. (Nature 334:364-366(1988)) disclosed that guanine-rich oligonucleotides can spontaneouslyself-assemble into four-stranded helices in vitro. Sen et al.(Biochemistry 31:65-70 (1992)) disclosed that these four-strandedcomplexes can further associate into superstructures composed of 8, 12,or 16 oligomers.

Marsh et al. (Biochemistry 33:10718-10724 (1994), and Nucleic AcidsResearch 23:696-700 (1995)) disclosed that some guanine-richoligonucleotides can also assemble in an offset, parallel alignment,forming long “G-wires”. These higher-order structures are stabilized byG-quartets that consist of four guanosine residues arranged in a planeand held together through Hoogsteen base pairings. According to Sen etal. (Biochemistry 31:65-70 (1992)), at least three contiguous guanineswithin the oligomer are critical for the formation of these higher orderstructures.

It has been suggested that four-stranded DNAs play a role in a varietyof biological processes, such as inhibition of HIV-1 integrase (Mazumderet al., Biochemistry 35:13762-13771 (1996)), formation of synapsisduring meiosis (Sen et al., Nature 334:364-366 (1988)), and telomeremaintenance (Williamson et al., Cell 59:871-880 (1989)); Baran et al.,Nucleic Acids Research 25:297-303 (1997)). It has been further suggestedthat controlling the production of guanine-rich quadruplexes might bethe key to controlling such biological processes. For example, U.S. Pat.No. 6,017,709 to Hardin et al. suggests that telomerase activity mightbe controlled through drugs that inhibit the formation of guaninequartets.

U.S. Pat. No. 5,888,739 to Pitner et al. discloses that G-quartet basedquadruplexes can be employed in an assay for detecting nucleic acids.Upon hybridization to a complementary oligonucleotide, the G-quartetstructure unfolds or linearizes, thereby increasing the distance betweendonor and acceptor moieties on different parts of the G-quartetstructure, resulting in a decrease in their interaction and a detectablechange in a signal (e.g., fluorescence) emitted from the structure.

U.S. Pat. No. 5,912,332 to Agrawal et al. discloses a method for thepurification of synthetic oligonucleotides, wherein the syntheticoligonucleotides hybridize specifically with a desired, full-lengtholigonucleotide and concomitantly form a multimer aggregate, such asquadruplex DNA. The multimer aggregate containing the oligonucleotide tobe purified is then isolated using size-exclusion techniques.

Despite the foregoing developments, a need has continued to exist tosystematically investigate and catalogue all specific interactionsbetween mixed base sequence nucleic acids and to create new, effectiveand rapid methods for producing and analyzing specific interactionbetween nucleic acids and/or nucleic acid analogues.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides a complex comprising: (1) a probe containing aheteropolymeric probe sequence of nucleic acids or nucleic acidanalogues; and (2) a target containing a heteropolymeric target sequenceof nucleic acids or nucleic acid analogues, wherein: (a) at least one ofthe probe and the target is purified or synthetic; and (b) theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by Watson-Crick complementary base interaction or by homologousbase interaction, provided that when the complex is a duplex and theheteropolymeric probe sequence is antiparallel to the heteropolymerictarget sequence, the heteropolymeric probe sequence is bonded to theheteropolymeric target sequence by homologous base interaction, andprovided that when the complex is a triplex, the complex is free ofrecombination proteins.

Also provided is a method for assaying a target, the method comprising:(1) providing a sample comprising the target containing aheteropolymeric target sequence of nucleic acids or nucleic acidanalogues; (2) providing a probe containing a heteropolymeric probesequence of nucleic acids or nucleic acid analogues; (3) providing ahybridization mixture comprising the target, the probe, water, and abuffer; (4) incubating the hybridization mixture for an incubation timeeffective to bind the heteropolymeric target sequence to theheteropolymeric probe sequence to provide a complex; and (5) detecting asignal correlated with binding affinity between the probe and the targetto assay the target, wherein the heteropolymeric probe sequence isbonded to the heteropolymeric target sequence by Watson-Crickcomplementary base interaction or by homologous base interaction,provided that when the complex is a duplex and the heteropolymeric probesequence is antiparallel to the heteropolymeric target sequence, theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base interaction, and provided that when thecomplex is a triplex, the complex is free of recombination proteins.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIGS. 1A, 1B, 1C, 2A, 2B, 3A, 3B, 4, 5A, 5B, 6, 7, 8, 9, 10A, 10B, 11A,11B, 12A, 12B, 13A and 13B are composite graphs of fluorescent intensityplotted as a function of wavelength for each sample analyzed;

FIGS. 14A, 14B and 14C are composite graphs of fluorescent intensityplotted as a function of time for each sample analyzed; and

FIG. 15 is a partially enlarged schematic view of a loop and stemembodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention flows from our elucidation of the specific bindingproperties of heteropolymeric nucleic acid strands. We have previouslydisclosed the specific binding of a heteropolymeric strand to duplexnucleic acid and the specific binding of duplex nucleic acid to otherduplex nucleic acid. We now disclose that heteropolymeric nucleic acids(and/or their analogues) can specifically bind to each other byhomologous base bonding as well as by Watson-Crick base interaction, andthat base bonding is not limited to strands having antiparalleldirectionality relative to each other. Thus, heteropolymeric nucleicacids (and/or their analogues) can specifically bind to each other withparallel or antiparallel directionality, wherein the bases bond byhomologous base bonding and/or Watson-Crick base bonding rules.

The invention is more than merely the disclosure of unorthodox bindingproperties of nucleic acids. The invention encompasses novel compounds,as well as methods for the analysis of nucleic acids, diagnosticmethods, therapeutic methods, prophylactic methods, gene therapy andgenetic engineering.

The invention encompasses novel duplex, triplex and quadruplex complexesof nucleic acids (and/or analogues thereof).

Nucleic acid strands have inherent directionality. The conventionalwisdom holds that strands of opposite directionality, i.e., which areantiparallel in their orientation to one another, can form a duplexthrough Watson-Crick complementary binding of their respective bases.

Certain duplexes according to the invention, on the other hand, comprisetwo strands of nucleic acid (and/or nucleic acid analogues) hybridizedin parallel relation to one another, wherein specific binding is eitherthrough homologous base pairing or Watson-Crick base pairing.Conventional wisdom holds that such duplexes do not exist, or at leastwould be extremely unstable due to, e.g., backbone irregularitiesnecessitated by the conformational requirements of parallel basebonding. Even more surprising is our discovery that under appropriatehybridization conditions, homologous bonding demonstrates specificityand stability rivaling that of Watson-Crick complementary antiparallelduplex.

The invention also encompasses duplexes containing two strands ofnucleic acid (and/or nucleic acid analogues) hybridized in antiparallelrelation to one another, wherein specific binding is through homologousbase pairing.

As used herein, the terms “Watson-Crick base pairing”, “complementarybase pairing” and the like are intended to define specific associationbetween opposing or adjacent pairs of nucleic acid and/or nucleic acidanalogue strands via matched bases (e.g., A:T; G:C and/or A:U). In thecontext of non-canonical complexes described herein, including parallelduplexes, parallel and antiparallel triplexes, and parallel andantiparallel quadruplexes, terms like “Watson-Crick base bonding” and“complementary base bonding” are intended to denote bonding between Aand T, A and U and/or G and C, but not necessarily in the edgewise,planar conformation first described by Watson and Crick. In addition tothe conventional binding motif first proposed by Watson and Crick (the“W-C motif”), and conformational variants thereof encompassed by theforegoing definition of Watson-Crick base bonding, the present inventionencompasses complexes formed by homologous base bonding. In homologousbase bonding, bases bond specifically with identical bases rather thancomplementary bases. Thus, in the “homologous motif”, homologous basepairs include A:A, G:G, C:C, T:T and U:U.

The binding by the bases of nucleic acid strands is affected orconditioned by a number of factors, particularly the binding potentialof the strands pursuant to either the W-C motif or homologous motif, andionic conditions (e.g., salt concentration and/or type). Saltyconditions tend to favor the formation of Watson-Crick bonding overhomologous bonding. Homologous motif quadruplexes are favored over W-Cmotif quadruplexes under identical buffer conditions probably becausethe localized environment can become relatively low-salt, based on thepresence of the charged backbones of the two duplex nucleic acids.

Each strand in a complex of the invention can comprise any sequence ofnucleobases and/or nucleobase analogues, provided the nucleobases arerelated to the nucleobases to which they are to specifically bind byeither the W-C motif or the homologous motif. Contrary to certainteachings of the prior art, the target and probe need not behomopolymeric to achieve binding, even in the case of triplex orquadruplex formation. Thus, in certain embodiments, the probenucleobases are arranged in a heteropolymeric probe sequence ofinterspersed purines and pyrimidines, and the target nucleobases arearranged in a target sequence at least partially complementary orpartially homologous to the probe sequence. For example, the probesequence can contain 25% to 75% purine bases and 75% to 25% pyrimidinebases in any order. Complexes of the invention can form fromheteropolymeric sequences, which as defined herein, means sequencescontaining at least one purine nucleobase or purine analogue and atleast one pyrimidine nucleobase or pyrimidine analogue in at least theirhybridizing segments. Heteropolymeric sequences preferably lackhomopolymeric fragments greater than 5 bases long. Other nucleobases arealso suitable for use in the invention, such as, e.g., syntheticanalogues of naturally occurring bases which have specific Watson-Crickand/or homologous binding affinities to other bases.

In addition to duplexes, complexes of the invention also includetriplexes and quadruplexes, wherein opposing heteropolymeric strands arelinked by Watson-Crick complementary bases or by homologous bases, andthe relative directionality of the bound sequences is parallel orantiparallel to one another.

A probe strand can specifically bind in the major or minor groove of adouble-stranded target. Further, the bases of a single-stranded probecan interact specifically with bases on one or both strands of adouble-stranded target. Similarly, the bases of each strand of adouble-stranded probe can interact specifically with bases on one orboth strands of a double-stranded target in quadruplex complexes of theinvention.

In certain triplex and quadruplex embodiments, each nucleobase binds toone or two other nucleobases. Thus, in addition to the traditionalduplex Watson-Crick base pairs and the duplex homologous base pairsdescribed above, such embodiments include the following Watson-Crickbase binding triplets: A:T:A, T:A:T, U:A:T, T:A:U, A:U:A, U:A:U, G:C:Gand/or C:G:C (including C⁺:G:C, and/or any other ionized species ofbase), and/or the following homologous base triplets: A:A:T, T:T:A,U:U:A, T:U:A, A:A:U, U:T:A, G:G:C and/or C:C:G (including C:C⁺:G, and/orany other ionized species of base).

Thus, in certain quadruplex embodiments wherein the probe is defined asa duplex of a first and a second strand and the target is defined as aduplex of a third and a fourth strand, it is believed that the bases ofthe first and third strands also bind to each other, in addition to: (a)the binding between opposing bases of the first and second strands; (b)the binding between opposing bases of the third and fourth strands; and(c) the binding between opposing bases of the second and fourth strands.

In certain embodiments of the triplex and quadruplex structures of theinvention, no binding sequence of bases is contiguous with anotherbinding sequence of bases. That is, there are at least three separatestrands. Although folded conformations and the like (e.g., hairpinturns, etc.) are within the scope of the invention, folded portions of asingle strand do not make the strand count more than once toward theminimum of three separate strands.

Complexes of the invention preferably do not rely on Hoogsteen bondingor G-G quartets for maintenance of the complex structure, althoughHoogsteen bonding and/or G-G quartets may be present. That is, complexesof the invention are preferably substantially free of Hoogsteen bonding,and substantially free of G-G quartets.

In certain complexes of the invention: (a) the probe is aheteropolymeric multistranded parallel complex; (b) bases of the probeare homologously bound in a first plane; (c) the target is aheteropolymeric multistranded parallel complex; and (d) bases of thetarget are homologously bound in a second plane. In certain othercomplexes of the invention: (a) the probe is a heteropolymericmultistranded parallel complex; (b) bases of the probe are boundpursuant to a Watson-Crick motif in a first plane; (c) the target is aheteropolymeric multistranded parallel complex; and (d) bases of thetarget are bound pursuant to a Watson-Crick motif in a second plane.

Each strand of the complex independently comprises a nucleic acid havinga deoxyribose phosphate or ribose phosphate backbone (e.g., DNA, RNA,mRNA, hnRNA, rRNA, tRNA or cDNA) or a nucleic acid analogue. Preferrednucleic acid analogues contain an uncharged or partially chargedbackbone (i.e., a backbone having a charge that is not as negative as anative DNA backbone), and include, e.g., PNA and LNA. Certainembodiments are free of PNA.

At least a portion of the complex is isolated, purified, artificial orsynthetic.

In embodiments, a portion of the complex is a PCR amplified product.

The complexes of the invention can be present in solution, on a solidsupport, in vitro, in vivo or in silico. The solid support can beelectrically conductive (e.g., an electrode) or non-conductive. Inaddition, the complexes can be optically mapped or sequenced after beingelongated, as taught in U.S. Pat. Nos. 6,147,198 and 5,720,928 toSchwartz.

Complexes of the invention can be provided by a method comprising: (a)providing a hybridization mixture comprising a target containing aheteropolymeric target sequence of nucleic acids or nucleic acidanalogues, a probe containing a heteropolymeric probe sequence ofnucleic acids or nucleic acid analogues, water, and a buffer; and (b)incubating said hybridization mixture for an incubation time effectiveto hybridize said heteropolymeric target sequence to saidheteropolymeric probe sequence to provide the complex.

The hybridization mixture can include any conventional medium known tobe suitable for preserving nucleotides. See, e.g., Sambrook et al.,“Molecular Cloning: A Lab Manual,” Vol. 2 (1989). For example, themedium can comprise nucleotides, water, buffers and standard saltconcentrations. When divalent cations are used exclusively to promotetriplex or quadruplex formation, chelators such as EDTA or EGTA shouldnot be included in the reaction mixtures.

Specific binding between complementary bases occurs under a wide varietyof conditions having variations in temperature, salt concentration,electrostatic strength, and buffer composition. Examples of theseconditions and methods for applying them are known in the art. Ourcopending U.S. patent application Ser. No. 09/885,731, filed Jun. 20,2001, discloses conditions particularly suited for use in thisinvention.

Unlike many Hoogsteen-type complexes, which are unstable or non-existentat pH levels above about 7.6, the complexes of the invention are stableover a wide range of pH levels, preferably from about pH 5 to about pH9.

Complexes of the invention can be provided for analytic, diagnostic,therapeutic and/or engineering purposes. The complexes can be used toanalyze, diagnose and/or treat conditions associated with infection byan organism or virus. The organism or virus can be quantitated, ifdesired.

Complexes of the invention can be formed under conventionalhybridization conditions, under triplex hybridization conditions, underquadruplex hybridization conditions or under conditions of in situhybridization. It is preferred that complexes be formed at a temperatureof about 2° C. to about 55° C. for about two hours or less. In certainembodiments, the incubation time is preferably less than five minutes,even at room temperature. Longer reaction times are not required, butincubation for up to 24 hours in most cases does not adversely affectthe complexes. The fast binding times of the complexes of the inventioncontrast with the much longer binding times necessary for Hoogsteenbound complexes.

The promoter in the hybridization medium is preferably an intercalatingagent or a cation, as disclosed in U.S. patent application Ser. No.09/613,263, filed Jul. 10, 2000. The intercalators are optionallyfluorescent. The intercalating agent can be, e.g., a fluorophore, suchas a member selected from the group consisting of YOYO-1, TOTO-1,YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyaninedimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1,BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, cyanine monomers,ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidiumderivatives, acridine, acridine orange, acridine derivatives,ethidium-acridine heterodimer, ethidium monoazide, propidium iodide,SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOX dyes and7-aminoactinomycin D.

Suitable cations include, e.g., monovalent cations, such as Na⁺(preferably at a concentration of 40 mM to 200 mM), K⁺ (preferably at aconcentration of 40 mM to 200 mM), and other alkali metal ions; divalentcations, such as alkaline earth metal ions (e.g., Mg⁺² and Ca⁺²) anddivalent transition metal ions (e.g., Mn⁺², Ni⁺², Cd⁺², Co⁺² and Zn⁺²);and cations having a positive charge of at least three, such as Co(NH₃)₆⁺³, trivalent spermidine and tetravalent spermine. Mn⁺² is preferablyprovided at a concentration of 10 mM to 45 mM. Mg⁺² is preferablyprovided at a concentration of 10 mM to 45 mM. Ni⁺² is preferablyprovided at a concentration of about 20 mM. In embodiments, Mg⁺² andMn⁺² are provided in combination at a concentration of 1 mM each, 2 mMeach, 3 mM each . . . 40 mM each (i.e., 1-40 mM each).

The amount of cation added to the medium in which the complex formsdepends on a number of factors, including the nature of the cation, theconcentration of probe, the concentration of target, the presence ofadditional cations and the base content of the probe and target. Thepreferred cation concentrations and mixtures can routinely be discoveredexperimentally. For triplexes, it is preferred to add cation(s) to themedium in the following amounts: (a) 10 mM-30 mM Mn⁺²; (b) 10 mM-20 mMMg⁺²; (c) 20 mM Ni⁺²; or (d) 1 mM-30 mM of each of Mn⁺² and Mg²⁺. Forquadruplexes, it is preferred to add cation(s) to the medium in thefollowing amounts: (a) 10 mM-45 mM Mn⁺²; (b) 10 mM-45 mM Mg⁺²; or (c) 10mM-40 mM of each of Mn⁺² and Mg⁺².

Although not required, other promoters include, e.g., single strandedbinding proteins such as Rec A protein, T4 gene 32 protein, E. colisingle stranded binding protein, major or minor nucleic acid groovebinding proteins, viologen and additional intercalating substances suchas actinomycin D, psoralen, and angelicin. Such facilitating reagentsmay prove useful in extreme operating conditions, for example, underabnormal pH levels or extremely high temperatures. Certain methods forproviding complexes of the invention are conducted in the absence ofprotein promoters, such as Rec A and/or other recombination proteins.

The invention provides a rapid, sensitive, environmentally friendly, andsafe method for assaying binding. The inventive assay can be used to,e.g., identify accessible regions in folded nucleotide sequences, todetermine the number of mismatched base pairs in a hybridizationcomplex, and to map genomes.

The inventive assay not only detects the presence of specificprobe-target binding, but also provides qualitative and quantitativeinformation regarding the nature of interaction between a probe andtarget. Thus, the invention enables the practitioner to distinguishamong a perfect match, a one base pair mismatch, a two base pairmismatch, a three base pair mismatch, a one base pair deletion, a twobase pair deletion and a three base pair deletion arising between asequence in the double-stranded probe or single-stranded probe and in asequence in the double-stranded or single-stranded target.

Embodiments of the invention comprise calibrating the measured signal(e.g., optical, fluorescence, chemiluminescence,electrochemiluminescence, electrical or electromechanical properties)for a first probe-target mixture against the same type of signalexhibited by other probes combined with the same target, wherein each ofthe other probes differs from the first probe by at least one base.

A calibration curve can be generated, wherein the magnitude of themeasured signal (e.g., fluorescent intensity) is a function of thebinding affinity between the target and probe. As the binding affinitybetween the target and a plurality of different probes varies with thenumber of mismatched bases, the nature of the mismatch(es) (e.g., A:Gvs. A:C vs. T:G vs. T:C, etc. in the W-C motif), the location of themismatch(es) within the complex, etc., the assay of the invention can beused to sequence the target.

In embodiments, the signal measured can be the fluorescent intensity ofa fluorophore included in the test sample. In such embodiments, thebinding affinity between the probe and target can be directly orinversely correlated with the intensity, depending on whether thefluorophore signals hybridization through signal quenching or signalamplification. Under selected conditions, the fluorescent intensitygenerated by intercalating agents can be directly correlated withprobe-target binding affinity, whereas the intensity of preferredembodiments employing a non-intercalating fluorophore covalently boundto the probe can be inversely correlated with probe-target bindingaffinity. The fluorescent intensity decreases for non-intercalatingfluorophores as the extent of matching (e.g., the amount of matches vs.mismatches and/or the types of mismatches) between the probe and targetincreases, preferably over a range inclusive of 0-2 mismatches and/ordeletions, more preferably over a range inclusive of 0-3 mismatchesand/or deletions.

The invention enables quantifying the binding affinity between probe andtarget. Such information can be valuable for a variety of uses,including designing antisense drugs with optimized bindingcharacteristics.

The assay of the invention is preferably homogeneous. The assay can beconducted without separating free probe and free target from thehybridization complex prior to detecting the magnitude of the measuredsignal. The assay does not require a gel separation step, therebyallowing a great increase in testing throughput. Quantitative analysesare simple and accurate. Consequently the binding assay saves a lot oftime and expense, and can be easily automated. Furthermore, it enablesbinding variables such as buffer, pH, ionic concentration, temperature,incubation time, relative concentrations of probe and target sequences,intercalator concentration, length of target sequences, length of probesequences, and possible cofactor (i.e., promoter) requirements to berapidly determined.

The assay can be conducted in, e.g., a solution within a well ormicrochannel, on an impermeable surface or on a biochip. In certainembodiments, the target is provided in the hybridization medium beforethe probe, and the probe is provided in dehydrated form prior torehydration by contact with the hybridization medium.

In certain embodiments, the inventive assay is conducted withoutproviding a signal quenching agent on the target or on the probe.

The invention obviates the need to denature the target prior toassaying. It is surprising that the inventors have been able tospecifically assay heteropolymeric triplexes and quadruplexes, whereinthe interaction between the probes and targets is based on Watson-Crickor homologous base interaction (at least in the sense that A binds to T(or U, in the case of RNA) and G binds to C), rather than the verylimited Hoogsteen model of complex hybridization of, e.g., Pitner etal., supra.

Suitable targets are preferably 8 to 3.3×10⁹ base pairs long, and can besingle or double-stranded.

Probes of the invention are preferably 2 to 75 bases long (morepreferably 5 to 30 bases long), and can be single or double-stranded.Thus, suitable probes for use in the inventive assay include, e.g.,ssDNA, RNA, ssPNA, LNA, dsDNA, dsRNA, DNA:RNA hybrids, dsPNA, PNA:DNAhybrids and other single and double-stranded nucleic acids and nucleicacid analogues having uncharged, partially-charged, sugar phosphateand/or peptide backbones. The length of the probe can be selected tomatch the length of the target.

The probe can be unfolded or folded. When the probe is folded, it ispreferred that the probe contains loop 20 and stem 22 adjacent to theloop, such as depicted in FIG. 15. Heteropolymeric probe sequence 24 islocated partially or entirely within stem 22 or loop 20. FIG. 15 showsthe most preferred location for heteropolymeric probe sequence 24,entirely within loop 20. This portion of loop 20 is enlarged to show anexemplary base sequence (A-T-G-C) of heteropolymeric probe sequence 24.

In embodiments wherein heteropolymeric probe sequence 24 is located inloop 20, it is preferred that binding of the heteropolymeric targetsequence (not shown) to the heteropolymeric probe sequence causes theopposing strands of stem 22 to separate, thereby opening loop 20. As theopposing strands separate, the distance between donor 26 and acceptor 28increases, resulting in a change in a detectable signal, which isparticularly useful for a method of detecting binding between theheteropolymeric probe sequence and the heteropolymeric target sequence.

In certain embodiments, donor 26 emits the signal and acceptor 28quenches the signal. In certain other embodiments, donor 26 transfersenergy to acceptor 28 and the acceptor emits the signal. Thus, donor 26and acceptor 28 can be, e.g., a fluorescence resonance energy transfer(FRET) pair or an electrochemiluminescent pair. Although donor 26 andacceptor 28 are schematically depicted in FIG. 15 as being differentfrom each other, it is possible for them to be identical to each other.Similarly, these embodiments of the invention are not limited to onlyone acceptor-donor pair placed at the free ends of stem 22. One or moreacceptor(s) and one or more donor(s) can be placed (e.g., via covalentbonding) throughout stem 22 in such “molecular beacon” embodiments. Thenumber of crosslinks 30 between adjacent sequences of stem 22 varies andcan be less or more than the five depicted in FIG. 15.

The use of certain single-stranded loop and stem structures as molecularbeacons to detect target nucleic acids is disclosed in, e.g., U.S. Pat.No. 5,925,517 to Tyagi et al.; Bonnet et al., Proc Natl Acad Sci USA1998 July 21; 95(15):8602-6; and Phys Rev Lett 2000 September11;85(11):2400-3. However, these references do not disclose or suggestthe use of molecular beacons to detect probe-target hybridizationaccording to the novel motifs of this invention. Embodiments containingthe heteropolymeric probe sequence in the stem can have enhancedspecificity for perfectly matched heteropolymeric target sequencesrelative to heteropolymeric probe sequences lacking an adjacent loop(possibly because of the enhanced stability of the resulting complex ofperfectly matched target and loop-containing probe). Some advantages ofloop and stem structures in conventional nucleic acid hybridizationstudies are suggested by Riccelli et al. (Nucleic Acids Research29:996-1004 (2001)) and U.S. Pat. No. 5,770,365 to Lane et al. Thepresent invention comprises the use of loop and stem probes containingthe novel complexes of the invention and/or adapted to form novelcomplexes of the invention when bound to the target. Thus, the loopand/or the stem of the probe can be completely or partiallysingle-stranded and/or double-stranded, and can even be triple-strandedor quadruple-stranded alone or after hybridization to the target. Theinstant invention does not require the use of radioactive probes, whichare hazardous, tedious and time-consuming to use, and need to beconstantly regenerated. Probes of the invention are preferably safe touse and stable for years. Accordingly, probes can be made or ordered inlarge quantities and stored.

The complex is preferably detected by a change in at least one label.The at least one label can be attached to the probe and/or the target,and/or can be free in the test medium. The at least one label cancomprise at least two moieties.

The label is preferably at least one member selected from the groupconsisting of a spin label, a fluorophore, a chromophore, achemiluminescent agent, an electro-chemiluminescent agent, aradioisotope, an enzyme, a hapten, an antibody and a labeled antibody.Preferably, the complex is detected by at least one emission from thelabel or by monitoring an electronic characteristic of the complex.

The labeled antibody can be, e.g., a labeled anti-nucleic acid/nucleicacid antibody, which can be labeled with a detectable moiety selectedfrom the group consisting of a fluorophore, a chromophore, a spin label,a radioisotope, an enzyme, a hapten, a chemiluminescent agent and anelectro-chemiluminescent agent.

The complex can be detected under at least one varied condition, such asdisclosed in U.S. patent application Ser. No. 09/490,273, filed Jan. 24,2000. Suitable varied conditions include, e.g., (a) a change innonaqueous components of the test medium, (b) a change in a pH of thetest medium, (c) a change in a salt concentration of the test medium,(d) a change of an organic solvent content of the test medium, (e) achange in a formamide content of the test medium, (f) a change in atemperature of the test medium, and (g) a change in chaotropic saltconcentration in the test medium. In addition, the varied condition canbe the application of a stimulus, such as, e.g., electric current (DCand/or AC), photon radiation (e.g., laser light), or electromagneticforce. The stimulus can be applied constantly or pulsed. Detection canbe accomplished through the use of a single varied condition, or througha combination of conditions varied serially.

The response of a characteristic of the complex in the test medium tothe varied condition or stimulus can be monitored to detect the complex.The characteristic can be, e.g., electrical conductance or Q (a resonantstructure of a transmission line or changes in phase or amplitude of asignal propagated in the transmission line in the test medium).

In embodiments, the detection method comprises: (a) detecting a signalfrom a label, wherein the signal is correlated to a binding affinitybetween said probe and said target; (b) varying a condition of a testmedium; (c) detecting a subsequent signal; and (d) comparing the signaland the subsequent signal. The varying and the detecting can be repeatedat least once or performed only once.

The label is preferably a fluorophore. Both intercalating andnon-intercalating fluorophores are suitable for use in the invention.The fluorophore can be free in solution, covalently bound to the probeand/or covalently bound to the target. When the fluorophore iscovalently bound to the probe, it is preferably bound to the probe ateither end. Preferred fluorescent markers include biotin, rhodamine,acridine and fluorescein, and other markers that fluoresce whenirradiated with exciting energy. Suitable non-intercalating fluorophoresinclude, e.g., alexa dyes, BODIPY dyes, biotin conjugates, thiolreactive probes, fluorescein and its derivatives (including the “cagedprobes”), Oregon Green, Rhodamine Green and QSY dyes (which quench thefluorescence of visible light excited fluorophores).

The excitation wavelength is selected (by routine experimentation and/orconventional knowledge) to correspond to this excitation maximum for thefluorophore being used, and is preferably 200 to 1000 nm. Fluorophoresare preferably selected to have an emission wavelength of 200 to 1000nm. In preferred embodiments, an argon ion laser is used to irradiatethe fluorophore with light having a wavelength in a range of 400 to 540nm, and fluorescent emission is detected in a range of 500 to 750 nm.

The assay of the invention can be performed over a wide variety oftemperatures, such as, e.g., from about 2 to about 60° C. Certain priorart assays require elevated temperatures, adding cost and delay to theassay. On the other hand, the invention can be conducted at roomtemperature or below (e.g., at a temperature below 25° C.).

The reliability of the invention is independent of guanine and cytosinecontent in either the probe or the target. In the traditional W-C motif,since G:C base pairs form three hydrogen bonds, while A:T base pairsform only two hydrogen bonds, target and probe sequences with a higher Gor C content are more stable, possessing higher melting temperatures.Consequently, base pair mismatches that increase the GC content of thehybridized probe and target region above that present in perfectlymatched hybrids may offset the binding weakness associated with amismatched probe.

The inventive assay is extremely sensitive, thereby obviating the needto conduct PCR amplification of the target. For example, it is possibleto assay a test sample having a volume of about 20 microliters, whichcontains about 10 femtomoles of target and about 10 femtomoles of probe.Embodiments of the invention are sensitive enough to assay targets at aconcentration of 5×10⁻⁹ M, preferably at a concentration of not morethan 5×10⁻¹⁰ M. Embodiments of the invention are sensitive enough toemploy probes at a concentration of 5×10⁹ M, preferably at aconcentration of not more than 5×10⁻¹⁰ M. It should go without sayingthat the foregoing values are not intended to suggest that the methodcannot detect higher concentrations.

The ratio of probe to target is preferably about 1:1 to about 1000:1.

Unlike certain prior art assays, the invention not only detects thepresence of hybridization (i.e., binding), but also provides qualitativeand quantitative information regarding the nature of binding between aprobe and target. Thus, the invention enables the practitioner to: (a)detect the presence of the target in the test medium; (b) detect allelicor heterozygous variance in the target; (c) quantitate the target; (d)detect an extent of complementarity (in the case of binding in the W-Cmotif) or homologousness (in the case of binding in the homologousmotif) between the probe and the target; and (e) detect haplotypes.

We have noticed that duplexes which complex parallel strands of nucleicacid containing complementary base sequences bind to form triplexes at adifferent rate and bind as a culmination of a very different processthan do bases in a double helix formed by nucleic acid strands ofopposite directionality. Strands of opposite directionality (i.e.,antiparallel strands) readily present regularly spaced bases in a planarorientation to the bases opposite with minimal backbone distortion.

The various complexes of the invention comprise a probe containing aheteropolymeric probe sequence of nucleobases and/or nucleobaseanalogues, and a target containing a heteropolymeric target sequence ofnucleobases and/or nucleobase analogues. The complex is synthetic orpurified in that at least one of either the probe or the target issynthetic or purified. The backbone of the probe is a deoxyribosephosphate backbone such as in DNA, or a peptide-like backbone such as inPNA, or is of some other uncharged or partially charged (negatively orpositively) moieties.

In certain embodiments, the probe and target are single-stranded and thecomplex is a duplex. When said probe and target are a duplex they haveparallel directionality with W-C complementary or homologous binding, orhave antiparallel directionality with homologous binding.

In other embodiments, either the probe or the target is single-strandedand the other of said probe or target is double-stranded and theresulting complex is a triplex. This complex can be free of PNA.

In certain embodiments, the triplex contains a heteropolymeric probesequence parallel to a heteropolymeric target sequence, wherein theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base binding or Watson-Crick complementary basebinding. In certain other embodiments, the heteropolymeric probesequence is antiparallel to the heteropolymeric target sequence and theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base binding or Watson-Crick complementary basebinding.

In certain embodiments of the triplex complex, the target includes afirst strand containing a heteropolymeric target sequence and a secondstrand containing a second heteropolymeric target sequence that isWatson-Crick complementary and antiparallel to the first heteropolymerictarget sequence. The heteropolymeric probe sequence is bonded to thefirst heteropolymeric target sequence by homologous base bonding and isalso bonded to the second heteropolymeric target sequence byWatson-Crick complementary base bonding.

In certain other embodiments of the triplex complex, the target includesa first strand containing a heteropolymeric target sequence and a secondstrand containing a second heteropolymeric target sequence that isWatson-Crick complementary and antiparallel to the first heteropolymerictarget sequence. The heteropolymeric probe sequence is bonded to thefirst heteropolymeric target sequence by Watson-Crick complementary basebonding and is also bonded to the second heteropolymeric target sequenceby homologous base bonding.

In certain embodiments, the probe and the target are double-stranded andthe resulting complex is a quadruplex. This complex can be free of PNA.

In certain embodiments, the quadruplex contains a heteropolymeric probesequence parallel or antiparallel to a heteropolymeric target sequence,wherein the heteropolymeric probe sequence is bonded to theheteropolymeric target sequence by homologous base binding orWatson-Crick complementary base binding. In such embodiments, thequadruplex complex contains a first probe strand containing saidheteropolymeric probe sequence and a second probe strand containing asecond heteropolymeric probe sequence that is complementary andantiparallel to the first probe sequence. The target includes a firsttarget strand containing a heteropolymeric target sequence and a secondtarget strand containing a second heteropolymeric target sequence thatis complementary and antiparallel to the first.

In such quadruplex embodiments, the heteropolymeric probe sequence canbond to the heteropolymeric target sequence by Watson-Crickcomplementary or homologous base binding and the heteropolymeric probesequence can optionally and additionally bond to the secondheteropolymeric target sequence by homologous or Watson-Crickcomplementary base binding, respectively. Thus, when the heteropolymericprobe sequence bonds to the heteropolymeric target sequence byhomologous base bonding, the heteropolymeric probe sequence optionallybonds to the second heteropolymeric target sequence by Watson-Crickcomplementary base bonding, and when the heteropolymeric probe sequencebonds to the heteropolymeric target sequence by Watson-Crickcomplementary base bonding, the heteropolymeric probe sequenceoptionally bonds to the second heteropolymeric target sequence byhomologous base bonding.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

Complementary sense and antisense 50-mer ssDNA target sequences, derivedfrom exon 10 of the human cystic fibrosis gene (Nature 380, 207 (1996))were synthesized on a DNA synthesizer (Expedite 8909, PerSeptiveBiosystems) and purified by HPLC. SsDNA oligonucleotides were dissolvedin ddH₂O and diluted to a concentration of 1 pmole/μl. Equimolar amountsof complementary oligonucleotides were heated at 95° C. for 10 min andallowed to anneal gradually in the presence of 10 mM Tris, pH 7.5, 1 mMEDTA and 100 mM NaCl, as the temperature cooled to 21° C. over 1.5hours. DsDNA oligonucleotides were diluted in ddH₂O at a concentrationof 1 pmole/μl.

The sequence for the sense strand of the wild-type target DNA (SEQ IDNO:1) was: 5′-TGG CAC CAT TAA AGA AAA TAT CAT CTT TGG TGT TTC CTA TGATGA ATA TA-3′.

The sequence for the antisense strand of the wild-type target DNA (SEQID NO:1) was: 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA TGA TAT TTT CTT TAATGG TGC CA-3′.

SEQ ID NO:2 was a 50-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a one base pair mutation(underlined) at amino acid position 507 at which the wild-type sensestrand sequence CAT was changed to CGT.

The sequence for the sense strand of SEQ ID NO:2 was:

5′-TGG CAC CAT TAA AGA AAA TAT CGT CTT TGG TGT TTC CTA TGA TGA ATATA-3′.

The sequence for the antisense strand of SEQ ID NO:2 was:

5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA CGA TAT TTT CTT TAA TGG TGCCA-3′.

SEQ ID NO:3 was a 50-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a consecutive two basepair mutation (underlined) at amino acid positions 506 and 507 at whichthe wild-type sense strand sequence CAT was changed to ACT.

The sequence for the sense strand of SEQ ID NO:3 was:

5′-TGG CAC CAT TAA AGA AAA TAT ACT CTT TGG TGT TTC CTA TGA TGA ATATA-3′.

The sequence for the antisense strand of SEQ ID NO:3 was:

5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA GTA TAT TTT CTT TAA TGG TGCCA-3′.

SEQ ID NO:4 was a 50-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a consecutive three basepair mutation (underlined) at amino acid positions 506 and 507 at whichthe wild-type sense strand sequence CAT was changed to ACG.

The sequence for the sense strand of SEQ ID NO:4 was:

5′-TGG CAC CAT TAA AGA AAA TAT ACG CTT TGG TGT TTC CTA TGA TGA ATATA-3′.

The sequence for the antisense strand of SEQ ID NO:4 was:

5′-TAT ATT CAT CAT AGG AAA CAC CAA AGC GTA TAT TTT CTT TAA TGG TGCCA-3′.

SEQ ID NO:5 was a 50-mer dsDNA target sequence modified from SEQ IDNO:1, wherein the percent GC content was changed from 30% to 52%.

The sequence for the sense strand of the wild-type target DNA (SEQ IDNO:5) was: 5′-GAG CAC CAT GAC AGA CAC TGT CAT CTC TGG TGT GTC CTA CGATGA CTC TG-3′.

The sequence for the antisense strand of the wild-type target DNA (SEQID NO:5) was: 5′-CAG AGT CAT CGT AGG ACA CAC CAG AGA TGA CAG TGT CTG TCATGG TGC TC-3′.

SEQ ID NO:6 was a 50-mer mutant dsDNA target sequence identical to SEQID NO:5, except for a one base pair mutation (underlined), at which thesense strand sequence CAT was changed to CGT.

The sequence for the sense strand of mutant SEQ ID NO:6 was: 5′-GAG CACCAT GAC AGA CAC TGT CGT CTC TGG TGT GTC CTA CGA TGA CTC TG-3′.

The sequence for the antisense strand of mutant SEQ ID NO:6 was: 5′-CAGAGT CAT CGT AGG ACA CAC CAG AGA CGA CAG TGT CTG TCA TGG TGC TC-3′.

SEQ ID NO:7 was a 50-mer mutant dsDNA target sequence identical to SEQID NO:5, except for a one base pair mutation (underlined), at which thesense strand sequence CTC was changed to CTT.

The sequence for the sense strand of mutant SEQ ID NO:7 was: 5′-GAG CACCAT GAC AGA CAC TGT CAT CTT TGG TGT GTC CTA CGA TGA CTC TG-3′.

The sequence for the antisense strand of mutant SEQ ID NO:7 was: 5′-CAGAGT CAT CGT AGG ACA CAC CAA AGA TGA CAG TGT CTG TCA TGG TGC TC-3′.

SEQ ID NO:8 was a 50-mer mutant dsDNA target sequence identical to SEQID NO:5, except for a consecutive two base pair mutation (underlined),at which the sense strand sequence CAT was changed to ACT.

The sequence for the sense strand of mutant SEQ ID NO:8 was: 5′-GAG CACCAT GAC AGA CAC TGT ACT CTC TGG TGT GTC CTA CGA TGA CTC TG-3′.

The sequence for the antisense strand of mutant SEQ ID NO:8 was: 5′-CAGAGT CAT CGT AGG ACA CAC CAG AGA GTA CAG TGT CTG TCA TGG TGC TC-3′.

SEQ ID NO:9 was a 47-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a consecutive three basepair deletion (indicated by three dots) at amino acid positions 507 and508 at which the wild-type sense strand sequence CTT is deleted.

The sequence for the sense strand of SEQ ID NO:9 was:

5′-TGG CAC CAT TAA AGA AAA TAT CAT . . . TGG TGT TTC CTA TGA TGA ATATA-3′.

The sequence for the antisense strand of SEQ ID NO:9 was:

5′-TAT ATT CAT CAT AGG AAA CAC CA . . . A TGA TAT TTT CTT TAA TGG TGCCA-3′.

SEQ ID NO:10 was a 50-mer mutant dsDNA target sequence identical to SEQID NO:5, except for a one base pair mutation (underlined), at which thesense strand sequence CAT was changed to CTT.

The sequence for the sense strand of mutant SEQ ID NO:10 was: 5′-GAG CACCAT GAC AGA CAC TGT CTT CTC TGG TGT GTC CTA CGA TGA CTC TG-3′.

The sequence for the antisense strand of mutant SEQ ID NO:10 was: 5′-CAGAGT CAT CGT AGG ACA CAC CAG AGA AGA CAG TGT CTG TCA TGG TGC TC-3′.

SEQ ID NO:11 was a 50-mer mutant dsDNA target sequence identical to SEQID NO:5, except for a one base pair mutation (underlined), at which thesense strand sequence CTC was changed to CCC.

The sequence for the sense strand of mutant SEQ ID NO:11 was: 5′-GAG CACCAT GAC AGA CAC TGT CAT CCC TGG TGT GTC CTA CGA TGA CTC TG-3′.

The sequence for the antisense strand of mutant SEQ ID NO:11 was: 5′-CAGAGT CAT CGT AGG ACA CAC CAG GGA TGA CAG TGT CTG TCA TGG TGC TC-3′.

The PNA probes were synthesized, HPLC purified and confirmed by massspectroscopy by Commonwealth Biotechnologies, Inc. (Richmond, Va., USA).PNA probes were first dissolved in 0.1% TFA (trifluoroacetic acid) to aconcentration of 10 mg/ml, and then diluted to 1 mg/ml by the additionof ddH₂O. Final PNA stock solutions were prepared in ddH₂O at aconcentration of 1 pmole/μl.

Probe No. 1 was a 15-mer PNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:1), overlapping amino acidpositions 505 to 510 (Nature 380, 207 (1996)). The directionality of theprobe was opposite or antiparallel to that of the sense strand in thetarget.

The sequence for Probe No. 1 (SEQ ID NO:12) was: 5′-H-CAC CAA AGA TGATAT-Lys-CONH₂-3′.

Probe No. 2 was a 15-mer PNA probe identical in sequence to Probe No. 1,but was of the same directionality, or parallel to that of the sensestrand in the dsDNA target.

The sequence for Probe No. 2 (SEQ ID NO:13) was: 5′-H-TAT AGT AGA AACCAC-Lys-CONH₂-3′.

The 15-mer ssDNA probes were synthesized and purified by HPLC as above.SsDNA probes were dissolved in ddH₂O at a concentration of 1 pmole/μl.

Probe No. 3 was a 15-mer ssDNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:5). The directionality of theprobe was opposite or antiparallel to that of the sense strand in thetarget.

The sequence for Probe No. 3 (SEQ ID NO:14) was: 5′-CAC CAG AGA TCACAG-3′.

Probe No. 4 was a 15-mer ssDNA probe identical in sequence to Probe No.3, but was of the same directionality, or parallel to that of the sensestrand in the dsDNA target.

The sequence for Probe No. 4 (SEQ ID NO:15) was: 5′-GAC AGT AGA GACCAC-3′.

Probe No. 5 was a 15-mer antiparallel ssDNA probe identical to Probe No.3, except it had an attached fluorescein moiety at the 5′ position.

The sequence for Probe No. 5 (SEQ ID NO:16) was: 5′-Flu-CAC CAG AGA TGACAG-3′.

Probe No. 6 was a 15-mer parallel ssDNA probe identical to Probe No. 4,except it had an attached fluorescein moiety at the 5′ position.

The sequence for Probe No. 6 (SEQ ID NO:17) was: 5′-Flu-GAC AGT AGA GACCAC-3′.

Probe No. 7 was a 15-mer ssDNA probe, with an attached fluoresceinmoiety at the 5′ position, designed to be completely complementary to a15 nucleotide segment of the sense strand of the 50-mer wild-type targetDNA (SEQ ID NO:1). The directionality of the probe was opposite orantiparallel to that of the sense strand in the target.

The sequence for Probe No. 7 (SEQ ID NO:18) was: 5′-Flu-CAC CAA AGA TGATAT-3′.

Probe No. 8 was a 15-mer ssDNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:1). The directionality of theprobe was antiparallel to that of the sense strand in the target.

The sequence for Probe No. 8 (SEQ ID NO:19) was: 5′-CAC CAA AGA TGATAT-3′.

Probe No. 9 and Probe No. 10 were 15-mer mutant ssDNA probes identicalin sequence to wild-type Probe No. 8, except for a one base mutation(underlined).

The sequence for Probe No. 9 (SEQ ID NO:20) was: 5′-CAC GAA AGA TGATAT-3′.

The sequence for Probe No. 10 (SEQ ID NO:21) was: 5′-CAC CAA ACA TGATAT-3′.

It is well known that ssDNA strands of mixed base sequence readily formssPNA:ssDNA duplexes on a Watson-Crick pairing basis when reacted witheither antiparallel or parallel synthesized ssPNA strands at roomtemperature. We have previously shown that such ssPNA:ssDNA complexescontaining perfectly matched sequences can reliably be distinguishedfrom ssPNA:ssDNA complexes containing a 1 bp mismatch when assayed inthe presence of the DNA intercalator, YOYO-1 (Molecular Probes, Eugene,Oreg., USA), and that the order of assembly of the PNA strand has asignificant bearing on its ability to specifically bind a ssDNA target.Example 1 compares the efficiency of formation of dsDNA duplexes whenwild-type or mutant ssDNA target sequences are reacted with Watson-Crickcomplementary antiparallel ssDNA probes or with homologous, that is tosay identical parallel, ssDNA probes.

The hybridization reaction mixtures giving rise to the data illustratedin FIG. 1A, each contained the following mixture: 2 pmoles of ssDNAtarget, 2 pmoles of ssDNA probe, 0.5×TBE and 500 nM of YOYO-1 in a finalvolume of 40 μl. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

In FIGS. 1B and 1C, the hybridization reaction mixtures (40 μl) eachcontained the following: 2 pmoles of ssDNA target, 2 pmoles of5′-fluorescein labeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 1 mMEDTA. The reaction mixtures were incubated at room temperature (21° C.)for 30 minutes or 90 minutes. Following incubation, each sample wasplaced into a quartz cuvette, irradiated with an argon ion laser beamhaving a wavelength of 488 nm and monitored for fluorescent emission.The maximum fluorescent intensities occurred at a wavelength of 525 nm,the emission wavelength for fluorescein. The intensity of fluorescentemission was plotted as a function of wavelength for each sampleanalyzed.

When the ssDNA Probe No. 3 was reacted with the 50-mer wild-type sensestrand of SEQ ID NO:5 or with the 50-mer mutant sense strand of SEQ IDNO:7 in the presence of YOYO-1, antiparallel complementary ssDNA:ssDNAduplexes were formed (FIG. 1A). The fluorescent intensity emitted by the1 bp T-G mismatched antiparallel complementary duplex (sense strand ofSEQ ID NO:7+Probe No. 3) was 56% lower than that obtained by theperfectly matched antiparallel complementary duplex (sense strand of SEQID NO:5+Probe No. 3).

When the ssDNA Probe No. 3 was reacted with the 50-mer wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1, theefficiency of parallel homologous ssDNA:ssDNA duplex formation was only3% lower than the efficiency of antiparallel complementary ssDNA:ssDNAduplex formation (FIG. 1A). This result was completely unanticipated.The 1 bp A-G mismatched parallel homologous duplex formed when the50-mer mutant antisense strand of SEQ ID NO:7 was reacted with the ssDNAProbe No. 3 in the presence of YOYO-1, produced a fluorescent emissionintensity that was 56% lower than that emitted by the perfectly parallelhomologous duplex (FIG. 1A). Control samples comprising each 50-merssDNA target plus 500 nM YOYO-1 exhibited levels of fluorescence whichranged from 91% to 92% lower than that observed with the perfectlymatched duplexes (FIG. 1A). The level of fluorescence emitted by the15-mer ssDNA Probe No. 3 plus 500 nM YOYO-1 was slightly greater thanthat produced by YOYO-1 alone. The shift in fluorescent emissionwavelength observed with the ssDNA targets and probe is typical ofYOYO-1's emission profile in the presence of ssDNA.

YOYO-1 facilitated DNA complex formation between a ssDNA probe and acomplementary base sequence in an antiparallel ssDNA target, or betweena ssDNA probe and an identical base sequence in a parallel ssDNA target,with similar efficacy, to allow differentiation between perfectlymatched complexes and those containing a 1 bp mismatch. In the parallelhomologous complexes, the 1 bp mismatch was a non-homologous base pair.

The comparative efficiency of antiparallel complementary and parallelhomologous dsDNA duplex formation was further examined using ssDNAtargets and ssDNA-F probes in the absence of complex promoting agentssuch as YOYO-1 or cations. When the ssDNA-F Probe No. 5 was incubatedfor 30 minutes in Tris buffer at room temperature with the 50-merwild-type sense strand of SEQ ID NO:5, the Watson-Crick complementaryantiparallel ssDNA:ssDNA-F duplexes were formed very efficiently,resulting in a 53% reduction in fluorescent emission compared to thatemitted by Probe No. 5 alone (FIG. 1B). By contrast, antiparallelcomplementary ssDNA:ssDNA-F complexes that contained a 1 bp T-G mismatch(sense strand of SEQ ID NO:7+Probe No. 5) were less stable, resulting inonly a 40% decrease in fluorescent emission compared to that emitted byProbe No. 5 alone after a 30 minute incubation (FIG. 1B).

Parallel homologous ssDNA:ssDNA-F complexes were formed when the ssDNAProbe No. 5 was reacted with the 50-mer wild-type antisense strand ofSEQ ID NO:5 or with the 50-mer mutant antisense strand of SEQ ID NO:7,generating fluorescent emission intensities that were 44% and 37% lower,respectively, than that emitted by ssDNA Probe No. 5 alone after a 30minute incubation (FIG. 1B). The avid formation of parallel homologousssDNA:ssDNA-F complexes in the absence of a promoting agent wascompletely unanticipated. The discrimination between signals emittedfrom perfectly matched duplexes and 1 bp mismatched duplexes in theabsence of complex promoting agents, was not as dramatic as thatobserved when YOYO-1 was present and served as the promoter andsignaling agent (compare FIGS. 1A and 1B). This was the case for bothantiparallel and parallel duplexes. Slightly less discrimination betweenperfectly matched and 1 bp mismatched DNA complexes was observed when aparallel homologous ssDNA target was used than when an antiparallelcomplementary ssDNA target was used to produce the ssDNA:ssDNA-Fcomplexes (FIG. 1B).

After a 90 minute incubation, Watson-Crick antiparallel dsDNA:ssDNA-Fcomplexes consisting of perfectly complementary sequences (sense strandof SEQ ID NO:5+Probe No. 5) or 1 bp T-G mismatched sequences (sensestrand of SEQ ID NO:7+Probe No. 5) produced a 39% and 30% decrease,respectively, in fluorescent emission intensity compared to that emittedby Probe No. 5 alone (FIG. 1C). Remarkably, parallel homologousssDNA:ssDNA-F complexes exhibited the same level of stability after 90minutes of incubation as did the Watson-Crick antiparallel ssDNA:ssDNA-Fcomplexes. The fluorescent intensities for a perfectly parallelhomologous duplex (antisense strand of SEQ ID NO:5+Probe No. 5) and a 1bp A-G mismatched parallel homologous duplex (antisense strand of SEQ IDNO:7+Probe No. 5) were 40% and 25% lower, respectively, than thatemitted by ssDNA Probe No. 5 alone after a 90 minute incubation (FIG.1C).

The mechanism of recognition and binding of the homologous bases in theparallel dsDNA duplexes is unknown at this time. Nevertheless,recognition and binding of parallel homologous ssDNA sequences occurredin a configuration which allowed the discrimination between perfectlymatched ssDNA:ssDNA complexes and those containing a 1 bp or 2 bpmismatch. In these parallel homologous complexes, the 1 bp mismatch wasa non-homologous base pair.

Example 2

In Example 1, the remarkable efficiency of parallel homologousssDNA:ssDNA duplex formation was demonstrated both in the presence of acomplex promoting agent such as YOYO-1 and in the absence of any complexpromoting agent. The recognition and binding of the homologous bases inthe parallel dsDNA duplexes was such as to allow easy discriminationbetween perfectly homologous base sequences and parallel homologoussequences that contained a 1 bp mismatch. These parallel homologous 1 bpmismatches were also clearly recognizable as mismatches based onWatson-Crick complementary recognition and binding rules. Example 2examines the recognition and binding efficiency of parallel homologousdsDNA duplexes that contain A-T or G-C base pairings, to determinewhether these Watson-Crick complementary pairings appear as mismatchesin a parallel homologous binding reaction.

Each hybridization reaction mixture (40 μl) contained the following: 2pmoles of ssDNA target, 2 pmoles of ssDNA probe, 0.5×TBE and 500 nM ofYOYO-1. The reaction mixtures were incubated at room temperature (21°C.) for 5 minutes, placed into a quartz cuvette, irradiated with anargon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescence was plotted as afunction of wavelength for each sample analyzed.

When the ssDNA Probe No. 3 (with a 53% GC content) was reacted with the50-mer wild-type antisense strand of SEQ ID NO:5 or with the 50-mermutant antisense strand of SEQ ID NO:10 in the presence of YOYO-1,parallel homologous ssDNA:ssDNA duplexes were formed (FIG. 2A). Thefluorescent intensity emitted by the 1 bp A-T mismatched parallelhomologous duplex (antisense strand of SEQ ID NO:10+Probe No. 3) was 72%lower than that obtained by the perfectly parallel homologous duplex(antisense strand of SEQ ID NO:5+Probe No. 3) (FIG. 2A). This dramaticdecrease in fluorescent emission by the parallel homologous duplexcontaining a 1 bp A-T, strongly suggested that the Watson-Crick A-Tbinding was hindered by the spatial and/or charge configuration imposedon the A and T bases when part of parallel homologous strands attemptingto achieve stable duplex. Control samples comprising each 50-mer ssDNAtarget plus 500 nM YOYO-1 exhibited levels of fluorescence which rangedfrom 96% to 97% lower than that observed with the perfectly matchedduplexes (FIG. 2A). The level of fluorescence emitted by the 15-merssDNA Probe No. 3 plus 500 nM YOYO-1 was slightly greater than thatproduced by YOYO-1 alone. The shift in fluorescent emission wavelengthobserved with the ssDNA targets and probe is typical of YOYO-1'semission profile in the presence of ssDNA.

Parallel homologous ssDNA:ssDNA duplexes were also formed when the50-mer wild-type antisense strand of SEQ ID NO:1 (with a 33% GC content)was reacted with the wild-type ssDNA Probe No. 8 or with the mutantssDNA Probes No. 9 and 10, in the presence of YOYO-1 (FIG. 2B). Thefluorescent intensities emitted by the 1 bp G-C mismatched parallelhomologous duplex (antisense strand of SEQ ID NO:1+Probe No. 9) and the1 bp C-G mismatched parallel homologous duplex (antisense strand of SEQID NO:1+Probe No. 10) were 67% and 66% lower, respectively, than thatobtained by the perfectly parallel homologous duplex (antisense strandof SEQ ID NO:1+Probe No. 8) (FIG. 2B). The configuration of theinteracting bases in the parallel homologous duplexes was unfavorablefor Watson-Crick complementary G-C binding, resulting in a decrease influorescent emission indicative of a 1 bp mismatch. Control samplesconsisting of the 50-mer ssDNA target plus 500 nM YOYO-1 or each of the15-mer ssDNA probes plus 500 nM YOYO-1 resulted in levels offluorescence that were slightly greater than that produced by YOYO-1alone (FIG. 2B).

Therefore, the interacting base pairs in parallel homologous dsDNAduplexes, formed in the presence of YOYO-1, adopt a configuration thatis unfavorable for binding between Watson-Crick complementary basepairs, resulting in such duplexes appearing to contain 1 bp mismatches.

We are led to envisage how mismatches in binding sequences, whetheroccurring as part of a hairpin or multistrand complex can causeenergetic and repeated motion as the base sequences try to achieve thestability of the ideal binding configuration under either binding motif.It is expected that binding strength of base pairs upstream ordownstream of nucleation sites, metal ions and other factors will have abearing on the attempts to achieve bonding.

Example 3

This example examines the efficiency of antiparallel homologousssDNA:ssDNA duplex formation facilitated by YOYO-1 or by monovalentcations.

The hybridization reactions, giving rise to the data illustrated in FIG.3A, each contained the following mixture: 2 pmoles of ssDNA target, 2pmoles of ssDNA probe, 0.5×TBE and 500 nM of YOYO-1 in a final volume of40 μl. The reaction mixtures were incubated at room temperature (21° C.)for 5 minutes, placed into a quartz cuvette, irradiated with an argonion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescent emission was plottedas a function of wavelength for each sample analyzed.

In FIG. 3B, the hybridization reaction mixtures (40 μl) each containedthe following: 2 pmoles of ssDNA target, 2 pmoles of 5′-fluoresceinlabeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 50 mM NaCl. Thereaction mixtures were incubated at room temperature (21° C.) forvarious lengths of time ranging from 1 minute to 60 minutes. Followingincubation, samples were placed into a quartz cuvette, irradiated withan argon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescent emission was plottedas a function of wavelength for each sample analyzed.

Incubation of ssDNA Probe No. 4 with the 50-mer wild-type antisensestrand of SEQ ID NO:5 in the presence of YOYO-1 resulted in antiparallelhomologous ssDNA:ssDNA complex formation (FIG. 3A). Although theefficiency of antiparallel homologous complex formation was only 65%that of conventional antiparallel complementary dsDNA formation (compareFIGS. 1A and 3A), recognition and binding of antiparallel homologousssDNA sequences did occur, facilitated by YOYO-1. This result wascompletely unanticipated. Furthermore, antiparallel homologousssDNA:ssDNA complexes comprising wild-type sequences were clearlydistinguished from those comprising 1 bp or 2 bp mismatches. Thefluorescent intensities emitted by the 1 bp A-G mismatched DNA complex(antisense strand of SEQ ID NO:7+Probe No. 4), the 1 bp C-T mismatchedDNA complex (antisense strand of SEQ ID NO:6+Probe No. 4), and theconsecutive 2 bp mismatched DNA complex (antisense strand of SEQ IDNO:8+Probe No. 4) were 25%, 65% and 71% lower, respectively, than thatobtained by the perfect antiparallel homologous complex (antisensestrand of SEQ ID NO:5+Probe No. 4) (FIG. 3A). As the degree of homologybetween the probe and target decreased, the level of fluorescentemission decreased. Control samples comprising each 50-mer ssDNA targetplus 500 nM YOYO-1 exhibited levels of fluorescence which ranged from88% to 90% lower than that observed with the perfectly matched complexes(FIG. 3A). The level of fluorescence emitted by the 15-mer ssDNA ProbeNo. 4 plus 500 nM YOYO-1 was slightly greater than that produced byYOYO-1 alone.

Antiparallel homologous ssDNA:ssDNA complex formation was furtherexamined using ssDNA targets and ssDNA-F probes both in the presence andabsence of 50 mM NaCl. After 15 minutes of incubation of ssDNA-F ProbeNo. 6 with the 50-mer wild-type antisense strand of SEQ ID NO:5 in thepresence of 50 mM NaCl, antiparallel homologous ssDNA:ssDNA-F complexeswere formed, as indicated by the 34% decrease in fluorescence observedcompared to that emitted by Probe No. 6 alone (FIG. 3B). The efficiencyof antiparallel homologous complex formation was 62% that ofantiparallel complementary complex formation following a 15 minuteincubation (data not shown). By contrast, antiparallel homologousssDNA:ssDNA-F complexes that contained a 1 bp A-G mismatch (antisensestrand of SEQ ID NO:7+Probe No. 6), a 1 bp C-T mismatch (antisensestrand of SEQ ID NO:6+Probe No. 6), a 1 bp A-T mismatch (antisensestrand of SEQ ID NO:10+Probe No. 6), and a consecutive 2 bp mismatch(antisense strand of SEQ ID NO:8+Probe No. 6), produced a 24%, 26%, 23%and a 13% decrease in fluorescence, respectively, compared to thatemitted by Probe No. 6 alone after a 15 minute incubation (FIG. 3B). Theconfiguration of the interacting bases in the antiparallel homologousduplexes was apparently unfavorable for Watson-Crick complementary A-Tbinding, resulting in a change in fluorescent emission indicative of a 1bp mismatch. Less antiparallel homologous complex formation occurredfollowing a 30 minute incubation in the presence of 50 mM NaCl (data notshown). No complex formation was evident after 45 minutes of incubation.Similar rates of antiparallel homologous complex formation and stabilitywere observed in Tris buffer without NaCl (data not shown).

Promoted by YOYO-1 or NaCl, recognition and binding of antiparallelhomologous ssDNA sequences occurred in a configuration which allowed thediscrimination between perfectly matched ssDNA:ssDNA complexes and thosecontaining a 1 bp or 2 bp mismatch. The interaction of the base pairs inthe antiparallel homologous duplex resulted in a conventionalWatson-Crick A-T base pair being destabilizing as a mismatch.

Example 4

This example demonstrates the efficiency of parallel complementaryssDNA:ssDNA complex formation promoted by monovalent cations. Thehybridization reaction mixtures (40 μl) each contained the following: 2pmoles of ssDNA target, 2 pmoles of 5′-fluorescein labeled ssDNA probe,10 mM Tris-HCl, pH 7.5, and 50 mM NaCl. The reaction mixtures wereincubated at room temperature (21° C.) for various lengths of timeranging from 1 minute to 60 minutes. Following incubation, samples wereplaced into a quartz cuvette, irradiated with an argon ion laser beamhaving a wavelength of 488 nm and monitored for fluorescent emission.The intensity of fluorescent emission was plotted as a function ofwavelength for each sample analyzed.

After a 15 minute incubation in the presence of 50 mM NaCl,ssDNA:ssDNA-F duplexes consisting of perfectly complementary sequences(sense strand of SEQ ID NO:5+Probe No. 6) formed readily, resulting in a41% decrease in fluorescent emission intensity compared to that emittedby Probe No. 6 alone (FIG. 4). This high efficiency of parallelcomplementary duplex formation was completely unexpected. By contrast,incompletely complementary ssDNA:ssDNA-F complexes containing a 1 bp T-Gmismatch (sense strand of SEQ ID NO:7+Probe No. 6), a 1 bp G-T mismatch(sense strand of SEQ ID NO:6+Probe No. 6), a 1 bp T-T mismatch (sensestrand of SEQ ID NO:10+Probe No. 6), and a consecutive 2 bp mismatch(sense strand of SEQ ID NO:8+Probe No. 6), generated an 18%, 20%, 10%and 16% decrease, respectively, in fluorescent emission intensitycompared to that exhibited by Probe No. 6 alone (FIG. 4).

Once formed in the presence of 50 mM NaCl, the perfectly matchedparallel complementary duplexes were very stable, resulting in a 40% and47% decrease in fluorescent emission after 30 minutes and 45 minutes ofincubation, respectively, compared to that emitted by Probe No. 6 alone(data not shown). The 1 bp and 2 bp mismatched parallel complementarycomplexes were much less stable after 30 minutes and 45 minutes ofincubation in the presence of 50 mM NaCl (data not shown). The rate andefficiency of parallel complementary ssDNA:ssDNA-F formation was verysimilar to that of antiparallel complementary ssDNA:ssDNA-F formationduring the first 45 minutes of incubation in the presence of 50 mM NaCl(data not shown). While antiparallel complementary complexes continuedto form easily after 60 minutes of incubation in 50 mM NaCl, no parallelcomplementary complex formation was evident at this time (data notshown).

NaCl facilitated DNA complex formation between a ssDNA-F probe and anantiparallel complementary ssDNA target, or between a ssDNA-F probe anda parallel complementary ssDNA target, with similar efficacy, to allowdifferentiation between perfectly matched complexes and those containinga 1 bp or 2 bp mismatch.

Example 5

Examples 1 to 4 demonstrated alternate base recognition and bindingmotifs occurring between antiparallel or parallel ssDNA probes, andcomplementary or homologous ssDNA targets to generate ssDNA:ssDNAduplexes, other than the conventional antiparallel Watson-Crickcomplementary dsDNA complexes. This example will show that bases arecapable of recognizing and interacting with both complementary andhomologous bases at the same time.

Samples of two pmoles of ssDNA Probe No. 3 were heated at 95° C. for 10minutes and allowed to cool to room temperature for 30 minutes in thepresence of various concentrations of a free base, resulting in ssDNAprobes containing conjugated bases. Duplicate samples of ssDNA Probe No.3 were similarly denatured and cooled in the absence of added free basesto generate non-conjugated ssDNA probes. Two pmoles of these conjugatedor non-conjugated ssDNA probes were then mixed with 2 pmoles of ssDNAtarget in the presence of 500 nM YOYO-1 and 0.5×TBE in a final reactionvolume of 40 μl. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nm,and monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

When the non-conjugated ssDNA Probe No. 3 was reacted with the 50-merwild-type sense strand of SEQ ID NO:5 or with the 50-mer mutant sensestrand of SEQ ID NO:7, in the presence of YOYO-1, antiparallelcomplementary ssDNA:ssDNA complexes were formed (FIG. 5A). Thefluorescent intensity emitted by the 1 bp T-G mismatched antiparallelcomplementary duplex (sense strand of SEQ ID NO:7+Probe No. 3) was 45%lower than that obtained by the perfectly matched antiparallelcomplementary duplex (sense strand of SEQ ID NO:5+Probe No. 3). Controlsamples comprising each 50-mer ssDNA target plus 500 nM YOYO-1 exhibitedlevels of fluorescence which ranged from 92% to 93% lower than thatobserved with the perfectly matched duplexes (FIG. 5A). The level offluorescence emitted by the 15-mer ssDNA Probe No. 3 plus 500 nM YOYO-1was slightly greater than that produced by YOYO-1 alone.

When the ssDNA Probe No. 3 was reacted with the 50-mer wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1, theefficiency of parallel homologous ssDNA:ssDNA duplex formation was 14%lower than the efficiency of antiparallel complementary ssDNA:ssDNAduplex formation (compare FIGS. 5A and 5B). The 1 bp A-G mismatchedparallel homologous duplex formed when the 50-mer mutant antisensestrand of SEQ ID NO:7 was reacted with the ssDNA Probe No. 3 in thepresence of YOYO-1, produced a fluorescent emission intensity that was47% lower than that emitted by the perfectly parallel homologous duplex(FIG. 5B).

The 15-mer ssDNA Probe No. 3 contains six adenine bases. Conjugation of2 pmoles of ssDNA Probe No. 3 with 3 pmoles of free thymine could resultin 25% of the complementary A or 100% of the homologous T within ProbeNo. 3 bound to the added thymine. Complementary A-T binding isenergetically preferred. Reaction of 2 pmoles of ssDNA Probe No. 3(conjugated with 3 pmoles of thymine) with 2 pmoles of the wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1 resulted indramatically enhanced parallel homologous ssDNA:ssDNA complex formation(FIG. 5B). Twenty-five percent conjugation of the ssDNA probe with 3pmoles of thymine increased parallel homologous complex formationbetween the perfectly homologous sequences by 78%. This augmentation ofparallel homologous complex formation can be linked to the ability ofthe adenines in Probe No. 3 to interact simultaneously with theconjugated complementary thymine bases, as well as with the homologousadenines in the ssDNA target. Moreover, interaction with availablecomplementary bases was not deleterious to the homologous bindingconfiguration adopted by the homologous bases and their neighbors.

By contrast, the efficiency of formation of parallel homologouscomplexes containing a 1 bp A-G mismatch (antisense strand of SEQ IDNO:7+Probe No. 3) were increased by 16% when Probe No. 3 was conjugated25% with thymine than when non-conjugated Probe No. 3 was used (FIG.5B). This corresponded to a 65% reduction in fluorescent emissionintensity for the 1 bp A-G mismatched parallel homologous complexcompared to that observed for the perfectly matched parallel homologouscomplex when the T-conjugated Probe No. 3 was used. Conjugation of thessDNA probe increased the specificity in discriminating betweenperfectly matched parallel homologous complexes and 1 bp mismatchedparallel homologous complexes.

Remarkably, perfectly matched antiparallel complementary ssDNA:ssDNAcomplex formation was enhanced by 48% when Probe No. 3 conjugated 25%with thymine was reacted with the sense strand of SEQ ID NO:5 in thepresence of YOYO-1 (FIG. 5A). The simultaneous interaction of an adeninein Probe No. 3 with the conjugated complementary thymine and thecomplementary T in the ssDNA target augmented formation of the perfectlymatched antiparallel complementary complex. Remarkably, formation of the1 bp T-G mismatched antiparallel complementary complex was veryinefficient when T-conjugated Probe No. 3 was used, resulting in an 88%decrease in fluorescent emission intensity compared to that generated bythe perfectly matched antiparallel complementary complex containingconjugated T (FIG. 5A). It is also remarkable that discriminationbetween perfectly matched and 1 bp mismatched antiparallel complementaryssDNA:ssDNA complexes was greatly enhanced by use of conjugated ssDNAprobes in the presence of YOYO-1.

Twenty-five percent conjugation of Probe No. 3 with cytosine orguanosine also increased the efficiency of both antiparallelcomplementary and parallel homologous ssDNA:ssDNA complex formation inthe presence of YOYO-1, as well as improved the specificity indifferentiation between perfectly matched complexes and 1 bp mismatchedcomplexes (data not shown).

Formation of ssDNA:ssDNA complexes comprising conjugated bases provesthat the bases in a sequence can recognize and interact specifically andsimultaneously with both complementary and homologous bases provided theconjugated base is a Watson-Crick complement to a base on the strandwhich binds specifically to another strand. The recognition and bindingconfigurations between bases in a ssDNA probe, conjugated bases andbases in a ssDNA target may be similar to the base configurations formedin antiparallel and parallel dsDNA:ssDNA complexes described herein.

Example 6

Example 6 demonstrates quadruplex DNA formation between dsDNA targetscontaining mixed base sequences and homologous dsDNA probes labeled withfluorescein. Quadruplex DNA formation is enhanced by the presence ofmonovalent cations added to the reaction. Complementary sense andantisense 15-mer ssDNA sequences were synthesized, purified by HPLC andannealed as above to generate 15-mer dsDNA probes. DsDNA probes werediluted in ddH₂O at a concentration of 1 pmole/μl.

Probe No. 11 was a 15-mer dsDNA probe with an attached fluoresceinmoiety at each 5′ position, and was designed to be completely homologousto a central 15 bp segment of the 50-mer wild-type target DNA (SEQ IDNO:5).

The sequence for the sense strand of Probe No. 11 (SEQ ID NO:22) was:5′-Flu-CTG TCA TCT CTG GTG-3′.

The sequence for the antisense strand of Probe No. 11 (SEQ ID NO:22)was: 5′-Flu-CAC CAG AGA TGA CAG-3′.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of target dsDNA, 4 pmoles of 5′-fluorescein labeled dsDNA ProbeNo. 11, 10 mM Tris-HCl, pH 7.5 and 100 mM KCl. The reaction mixtureswere incubated at room temperature (21° C.) for 1 hour, without priordenaturation. Samples were placed into a quartz cuvette, irradiated withan argon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The maximum fluorescent intensities occurred at awavelength of 525 nm, the emission wavelength for fluorescein. FIG. 6shows the intensity of fluorescence plotted as a function of wavelengthfor each sample analyzed.

In the absence of KCl, no binding between the dsDNA targets and ProbeNo. 11 was detected, resulting in similar fluorescent intensitiesobserved when wild-type dsDNA target (SEQ ID NO:5) or mutant dsDNAtarget (SEQ ID NO:7) were mixed with dsDNA Probe No. 11 or when dsDNAProbe No. 11 was present alone (data not shown).

After a 1 hour incubation at 21° C. in the presence of 100 mM KCl,dsDNA:dsDNA-F quadruplexes consisting of perfectly homologous sequenceson dsDNA target (SEQ ID NO:5) and dsDNA Probe No. 11 formed readily,resulting in a 62% decrease in the intensity of fluorescent emissioncompared to that emitted by dsDNA Probe No. 11 alone (labeled dsDNA-F)(FIG. 6). In contrast, incompletely homologous dsDNA:dsDNA-Fquadruplexes (SEQ ID NO:7+Probe No. 11), containing a 1 base pairmismatch were less stable in these reaction conditions, yielding only an18% decrease in fluorescent intensity compared to that exhibited bydsDNA Probe No. 11 alone.

The presence of monovalent cations, such as K⁺, at specificconcentrations was sufficient to allow quadruplex formation betweendsDNA targets and dsDNA probes labeled with fluorescein in the absenceof prior denaturation. Quadruplex formation occurred on the basis ofhomologous base pair affinities, with a measurable and significantlygreater amount of quadruplex formation between fully homologous duplexstrands. Moreover, the reaction occurred at room temperature within just1 hour of incubation at a ratio of probe to target of 10 to 1, usingnatural dsDNA. The dsDNA targets and dsDNA probe used in this examplewere homologous, contained 53% GC content, and did not containhomopurine or homopyrimidine stretches on any DNA strand. The assay ofthe invention was able to identify perfectly homologous dsDNA sequencesand those containing a pair of mismatched bases, using a dsDNA probe.

Example 7

The quadruplex DNA assays performed in Example 6 were facilitated by theaddition of monovalent cations in the reaction mixtures. The specificityof the assay was further examined utilizing divalent cations tofacilitate quadruplex DNA formation with dsDNA targets and dsDNA-Fprobes possessing 53% GC content.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of target dsDNA, 4 pmoles of 5′-fluorescein labeled dsDNA ProbeNo. 11, 10 mM Tris-HCl, pH 7.5, 20 mM MnCl₂ and 20 mM MgCl₂. Thereaction mixtures were incubated at room temperature (21° C.) for 1hour, without prior denaturation. Samples were placed into a quartzcuvette, irradiated with an argon ion laser beam having a wavelength of488 nm and monitored for fluorescent emission. FIG. 7 shows theintensity of fluorescence plotted as a function of wavelength for eachsample analyzed.

When dsDNA-F Probe No. 11 (with a 53% GC content) was incubated with the50-mer wild-type dsDNA target (SEQ ID NO:5) or the mutant dsDNA target(SEQ ID NO:7) in the presence of 20 mM MnCl₂ and 20 mM MgCl₂,quadruplexes were formed at room temperature under non-denaturingconditions. While perfectly homologous DNA quadruplexes yielded themaximum decrease in fluorescent intensity, a 34% decrease, the lessfavourable dsDNA:dsDNA-F quadruplexes containing a 1 bp mismatch (SEQ IDNO:7+Probe No. 11) produced a fluorescent intensity that was about thesame as that observed with dsDNA Probe No. 11 alone (FIG. 7).

The presence of divalent cations such as Mn⁺² and Mg⁺² facilitatedquadruplex formation under non-denaturing conditions to allow accuratediscrimination between fully homologous dsDNA target and dsDNA probequadruplexes, and quadruplex sequences containing a pair of bases whichare mismatched.

Example 8

The quadruplex DNA assays performed in Examples 6 and 7 were facilitatedby the addition of either monovalent cations or divalent cations in thereaction mixtures. The next Example demonstrates the specificity of thehomologous quadruplex DNA assay when the DNA intercalator, YOYO-1, isemployed.

Complementary sense and antisense 15-mer ssDNA sequences weresynthesized, purified by HPLC and annealed as above to generate 15-merdsDNA probes. DsDNA probes were diluted in ddH₂O at a concentration of 1pmole/μl.

Probe No. 12 was a 15-mer dsDNA probe identical in sequence to Probe No.11, but without the attached 5′ fluorescein moieties.

The sequence for the sense strand of Probe No. 12 (SEQ ID NO:23) was:5′-CTG TCA TCT CTG GTG-3′.

The sequence for the antisense strand of Probe No. 12 (SEQ ID NO:23)was: 5′-CAC CAG AGA TGA CAG-3′.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of dsDNA target, 4 pmoles of dsDNA Probe No. 12, 0.5×TBE and 100nM of YOYO-1. The reaction mixtures were incubated at 21° C. for 5minutes, placed into a quartz cuvette, irradiated with an argon ionlaser beam having a wavelength of 488 nm and monitored for fluorescentemission. The intensity of fluorescent emission was plotted as afunction of wavelength for each sample analyzed.

The fluorescent intensities observed when no target or probe was present(YOYO-1 only) are shown in FIG. 8. FIG. 8 also shows the fluorescentintensities observed when the reaction mixtures combined dsDNA Probe No.12 with wild-type 50-mer dsDNA target (SEQ ID NO:5) which containedhomologous sequences, or with four other dsDNA targets which, but forone mismatched pair of bases, contained sequences which were homologousto the base sequences in the dsDNA Probe No. 12. Homologous wild-typedsDNA target (SEQ ID NO:5) when present in the reaction mixture with thedsDNA Probe No. 12 produced the greatest fluorescent intensity.Mismatched dsDNA targets when incubated with dsDNA Probe No. 12 in thereaction mixture yielded lesser fluorescent intensity values rangingfrom 20% less for dsDNA target (SEQ ID NO:10) to 80% less for dsDNAtarget (SEQ ID NO:11), compared to that achieved by perfectly matchedquadruplexes (FIG. 8).

It was observed that homologous quadruplexes, stabilized by YOYO-1intercalation, formed more readily between a dsDNA target and a dsDNAprobe when that probe contained perfectly homologous sequences, thanwhen there was a single pair of bases which were not homologous, that isto say identical, to a pair of bases in the dsDNA target. The quadruplexcomplexes described in the foregoing three examples are referred to byus as mirror homologous.

Example 9

In this example, 50-mer dsDNA targets were exposed to a 53% GC 15-merdsDNA probe (Probe No. 13), wherein Watson-Crick complementarity existsbetween bases of the strands of the probe and proximal bases of thestrands of the target when the major groove of one duplex is placed inthe minor groove of the other duplex. The sequences of bases in theduplex probe are not homologous but are inverted in relation to those inthe duplex target. The duplexes, when nested major groove into minorgroove, are parallel to one another, and referred to by us as nestedcomplementary.

Complementary sense and antisense 15-mer ssDNA sequences weresynthesized, purified by HPLC and annealed as above to generate 15-merdsDNA probes. DsDNA probes were diluted in ddH₂O at a concentration of 1pmole/μl.

The sequence for the sense strand of Probe No. 13 (SEQ ID NO:24) was:5′-GAC AGT AGA GAC CAC-3′.

The sequence for the antisense strand of Probe No. 13 (SEQ ID NO:24)was: 5′-GTG GTC TCT ACT GTC-3′.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of target dsDNA, 4 pmoles of dsDNA Probe No. 13, 0.5×TBE and 100nM of YOYO-1. The reaction mixtures were incubated at room temperature(21° C.) for 5 minutes, placed in a quartz cuvette, irradiated with anargon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescent emission was plottedas a function of wavelength for each sample analyzed.

FIG. 9 illustrates that in the absence of prior denaturation, thehighest fluorescent intensities were achieved when the wild-type 50-merdsDNA target (SEQ ID NO:5) was reacted with the 15-mer dsDNA Probe No.13, which was a perfect match on a nested complementary basis to thedsDNA target (SEQ ID NO:5). The fluorescent intensity is indicative ofDNA binding taking place, in this case quadruplex formation between thedsDNA target and the nested complementary dsDNA probe.

Mutant dsDNA targets which were mismatched with the duplex probe by asingle pair of bases when matching was assessed on the inverted homologybasis of nested complementarity, formed measurably fewer quadruplexcomplexes with the dsDNA probe, than did the fully complementarywild-type dsDNA target. The various mismatches, which were assayed on amirror homologous basis in Example 8 were assayed on a nestedcomplementary basis in this example.

As shown in FIG. 9, the fluorescent intensities produced by thequadruplexes formed with the 1 bp mismatched dsDNA targets plus dsDNAProbe No. 13, ranged from 8% to 16% less than that achieved by perfectlymatched quadruplexes (SEQ ID NO:5+Probe No. 13).

Greater discrimination in fluorescence was observed between perfectlyhomologous and partially homologous quadruplexes in Example 8. Thissuggests that fully complementary or 1 base pair mismatched nestedcomplementary dsDNA probes bind less discriminately to dsDNA targetsthan do mirror homologous dsDNA probes, which bind with greaterspecificity.

This example shows that Watson-Crick quadruplex binding between nestedcomplementary DNA duplexes readily occurs in the presence of YOYO-1.

Example 10

Example 10 demonstrates that the assay of the invention can discriminatebetween perfectly matched, Watson-Crick complementary dsDNA:ssPNAcomplexes and dsDNA:ssPNA complexes containing 1 bp, 2 bp and 3 bpmismatches when a cationic decondensing agent, such as the DNAintercalator, YOYO-1 is present.

Each hybridization reaction mixture (40 μl) contained the following: 2pmoles of target dsDNA, 2 pmoles of ssPNA probe, 0.5×TBE and 500 nM ofYOYO-1. The reaction mixtures were incubated at room temperature (21°C.) for 5 minutes, placed into a quartz cuvette, irradiated with anargon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescence was plotted as afunction of wavelength for each sample analyzed.

The fluorescent intensities observed when no DNA or PNA was present(YOYO-1 only), or when wild-type SEQ ID NO:1, mutant SEQ ID NO:2 ormutant SEQ ID NO:3 were reacted with antiparallel PNA Probe No. 1 orparallel PNA Probe No. 2 are shown in FIGS. 10A and 10B, respectively.DsDNA:ssPNA complexes consisting of perfectly complementary sequences(SEQ ID NO:1+Probe No. 1) allowed maximum interaction between YOYO-1 andthe complexes, yielding the highest fluorescent intensities (FIG. 10A).The fluorescent intensities for a one base pair mismatched dsDNA:ssPNAcomplex (SEQ ID NO:2+Probe No. 1) and a two base pair mismatcheddsDNA:ssPNA complex (SEQ ID NO:3+Probe No. 1) was 97% and 99% lower,respectively, than the perfectly matched dsDNA:ssPNA complex (FIG. 10A).Similarly, when parallel PNA Probe No. 2 was bound to the target dsDNAsequences, the one and two base pair mismatched dsDNA:ssPNA complexesexhibited fluorescent intensities that were 92% and 97% lower,respectively, than the perfectly complementary dsDNA:ssPNA complexes(SEQ ID NO:1+Probe No. 2) (FIG. 10B). Three base pair mismatcheddsDNA:ssPNA complexes consisting of SEQ ID NO:4 and Probe No. 1, or SEQID NO:4 and Probe No. 2 produced fluorescent intensities that were 99%and 97% lower, respectively, than the perfectly matched dsDNA:ssPNAcomplexes (data not shown). Control samples comprising 50-mer dsDNAtargets plus 500 nM YOYO-1 exhibited levels of fluorescence which wereat or below the level of fluorescence observed with 3 bp mismatchedcomplexes (data not shown). The level of fluorescence emitted by eitherssPNA probe plus 500 nM YOYO-1 together was identical to that emitted byYOYO-1 alone (data not shown). As the degree of mismatch between theprobe and the target increased, the level of interaction of YOYO-1 withthe mismatched complexes diminished. Hence the intensity of fluorescentemission decreased. This relationship held whether or not anantiparallel or parallel PNA probe was used. The characteristic level offluorescence emitted by each complex was monitored over time and wasstable between 5 minutes and 24 hours.

Interestingly, when 15-mer target dsDNA sequences were reacted with15-mer PNA probe sequences, larger differences in fluorescent emissionwere observed between perfectly matched complexes and 1 or 2 bpmismatched complexes when parallel PNA probes were used, than whenantiparallel PNA probes were used (data not shown).

Therefore, the fluorescent intensity assay measuring dsDNA:ssPNA complexformation is able to distinguish between wild-type sequences and thosecontaining 1 bp, 2 bp or 3 bp mutations, without prior denaturation ofthe duplex DNA target.

Example 11

The specificity of the assay measuring triplex formation promoted byYOYO-1 was further investigated by reacting wild-type and mutant dsDNAtargets of mixed base sequence with antiparallel and parallel ssDNAprobes in the absence of prior denaturation of dsDNA targets.

Each hybridization reaction mixture (40 μl) contained the following: 2pmoles of target dsDNA, 2 pmoles of ssDNA probe, 0.5×TBE and 500 nM ofYOYO-1. The reaction mixtures were incubated at room temperature (21°C.) for 5 minutes, placed into a quartz cuvette, irradiated with anargon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescence was plotted as afunction of wavelength for each sample analyzed.

When the antiparallel ssDNA Probe No. 3 (with a 53% GC content) wasreacted with the 50-mer wild-type dsDNA target (SEQ ID NO:5) and mutantdsDNA targets (SEQ ID NO:6 and SEQ ID NO:8), dsDNA:ssDNA complexes wereformed at room temperature under non-denaturing conditions (FIG. 11A).While perfectly matched DNA complexes emitted the highest fluorescentintensities, incompletely complementary complexes with a 1 bp mismatch(SEQ ID NO:6+Probe No. 3) and a consecutive 2 bp mismatch (SEQ IDNO:8+Probe No. 3) produced fluorescent intensities that were 63% and 95%lower, respectively, than that observed with the perfectly matchedsequences (FIG. 11A). The level of fluorescence diminished as the degreeof mismatch between the probe and target increased. The characteristicfluorescent intensity exhibited by each complex was monitored over timeand was stable between 5 minutes and 24 hours. Control samplescomprising 50-mer dsDNA targets plus 500 nM YOYO-1 exhibited levels offluorescence which were below the level of fluorescence observed with 2bp mismatched DNA complexes (data not shown). The level of fluorescencegenerated by the ssDNA probe plus 500 nM YOYO-1 was identical to thatachieved by YOYO-1 alone (data not shown). Very similar results wereobtained when 15-mer antiparallel ssDNA probes were reacted withwild-type or mutant 50-mer dsDNA targets having 33% GC and 73% GCcontents under the same reaction conditions, demonstrating thereliability of the dsDNA:ssDNA complex formation assay utilizingantiparallel ssDNA probes, independent of the percent GC content of thessDNA probes and dsDNA targets (data not shown).

Similarly, in the presence of YOYO-1, dsDNA:ssDNA complexes were formedwhen the parallel ssDNA Probe No. 4 was reacted with the 50-merwild-type dsDNA target (SEQ ID NO:5) and mutant dsDNA targets (SEQ IDNO:6 and SEQ ID NO:8). The fluorescent intensities for a 1 bp mismatchedDNA complex (SEQ ID NO:6+Probe No. 4) and a consecutive 2 bp mismatchedDNA complex (SEQ ID NO:8+Probe No. 4) were 48% and 65% lower,respectively, than that obtained by the perfectly matched sequences(FIG. 11B). As the degree of mismatch between the probe and targetincreased, the level of fluorescent emission decreased. Slightly lessdiscrimination between perfectly matched and mismatched DNA complexeswas observed when a parallel ssDNA probe was used than when anantiparallel ssDNA probe was used to generate the dsDNA:ssDNA complexes.

YOYO-1 facilitated DNA complex formation between an antiparallel ssDNAprobe and dsDNA targets, and between a parallel ssDNA probe and dsDNAtargets, to allow differentiation between perfectly matched complexesand those containing 1 bp or 2 bp mismatches, without the requirementfor prior denaturation of dsDNA targets.

Example 12

The complexes formed in Examples 10 and 11 were stabilized by the DNAintercalator, YOYO-1 present in the reaction mixtures. The specificityof the assay was further examined utilizing divalent cations to promoteand stabilize complex formation with dsDNA targets and ssDNA-F probes.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of target dsDNA, 4 pmoles of 5′-fluorescein labeled ssDNA probe,10 mM Tris-HCl, pH 7.5, and 1 mM to 20 mM each of MgCl₂ and MnCl₂. Thereaction mixtures were incubated at room temperature (21° C.) forvarious lengths of time ranging from 1 minute to 2 hours, without priordenaturation of dsDNA targets. Following incubation, samples were placedinto a quartz cuvette, irradiated with an argon ion laser beam having awavelength of 488 nm and monitored for fluorescent emission. The maximumfluorescent intensities occurred at a wavelength of 525 nm, the emissionwavelength for fluorescein. The intensity of fluorescent emission wasplotted as a function of wavelength for each sample analyzed.

When the antiparallel ssDNA-F Probe No. 5 was incubated for 1 hour withthe 50-mer wild-type dsDNA target (SEQ ID NO:5) in the presence of 15 mMMgCl₂ and 15 mM MnCl₂, perfectly complementary dsDNA:ssDNA-F complexeswere formed very efficiently, generating a 74% decrease in fluorescencecompared to that achieved by Probe No. 5 alone (FIG. 12A). By contrast,dsDNA:ssDNA-F complexes that contained a 1 bp T-G mismatch (SEQ IDNO:7+Probe No. 5) were much less stable in the presence of 15 mM MgCl₂and 15 mM MnCl₂, yielding a 15% decrease in fluorescence compared tothat emitted by Probe No. 5 alone after a 1 hour incubation (FIG. 12A).When Probe No. 5 (containing a 53% GC content) was reacted with thedsDNA target SEQ ID NO:9 (containing a 33% GC content), a 3% increase influorescence was observed compared to that obtained by Probe No. 5 alone(FIG. 12A), indicative of no DNA complex formation. This result wasexpected considering this probe and target combination would result in a5 bp mismatch.

In the presence of 10 mM MgCl₂ and 10 mM MnCl₂, the dsDNA:ssDNA-Fcomplexes possessing a 53% GC content and containing perfectlycomplementary sequences (SEQ ID NO:5+Probe No. 5) or a 1 bp T-G mismatch(SEQ ID NO:7+Probe No. 5) generated fluorescent intensities that were68% and 20% lower, respectively, after an 1 hour incubation, and 76% and16% lower, respectively, after a 30 minute incubation, than that emittedby Probe No. 5 alone (data not shown). The addition of 5 mM MgCl₂ and 5mM MnCl₂ (or lower concentrations) was insufficient to allow complexformation between the antiparallel ssDNA-F Probe No. 5 and all dsDNAtargets tested following a 1 hour incubation (data not shown).

DsDNA:ssDNA complexes were also formed when the parallel ssDNA Probe No.6 was reacted with the 50-mer wild-type dsDNA target (SEQ ID NO:5) andmutant dsDNA target (SEQ ID NO:7). In this case DNA complex formationwas promoted with much lower concentrations of MgCl₂ and MnCl₂ (i.e. 1-5mM each) requiring shorter incubation periods. Incubation in thepresence of 1 mM MgCl₂ and 1 mM MnCl₂, or 2 mM MgCl₂ and 2 mM MnCl₂ for15 minutes was sufficient to generate DNA complexes (data not shown).The fluorescent intensities for a perfectly matched DNA complex (SEQ IDNO:5+Probe No. 6) and a 1 bp mismatched DNA complex (SEQ ID NO:7+ProbeNo. 6) were 29% and 6% lower, respectively, than that obtained byparallel ssDNA Probe No. 6 alone in the presence of 3 mM MgCl₂ and 3 mMMnCl₂ after a 45 minute incubation (FIG. 12B).

Although DNA complexes formed readily at 10 mM MgCl₂ and 10 mM MnCl₂after a 1 hour incubation, no discrimination between perfectly matchedand mismatched complexes was observed when a parallel ssDNA probe wasused. Concentrations above 15 mM MgCl₂ and 15 mM MnCl₂ were inhibitoryfor DNA complex formation with a parallel ssDNA probe (data not shown).

The addition of salt bridging, condensing agents such as divalentcations promoted DNA complex formation between non-denatured dsDNAtargets and fluorescently-labeled antiparallel or parallel ssDNA probes,to allow accurate and reliable discrimination between perfectlycomplementary sequences and those containing 1 bp mutations. Thereactions occurred at room temperature within 15-60 minutes ofincubation at a ratio of probe to target of 10 to 1. The dsDNA targetsand ssDNA probes did not contain homopurine or homopyrimidine stretchesof DNA. Despite the presence of 5 pyrimidine bases interspersed withinthe 15 nucleotide ssDNA probes, DNA complexes formed readily in asequence specific manner.

Example 13

The utility of probes of varying directionality was also evaluated whenmonovalent cations were employed to promote and stabilize complexformation with dsDNA targets.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of target dsDNA, 4 pmoles of 5′-fluorescein labeled ssDNA probe,10 mM Tris-HCl, pH 7.5, and 10 mM to 150 mM NaCl. The reaction mixtureswere incubated at room temperature (21° C.) for various lengths of timeranging from 1 minute to 2 hours, without prior denaturation of dsDNAtargets. Following incubation, samples were placed into a quartzcuvette, irradiated with an argon ion laser beam having a wavelength of488 nm and monitored for fluorescent emission. The maximum fluorescentintensities occurred at a wavelength of 525 nm, the emission wavelengthfor fluorescein. The intensity of fluorescent emission was plotted as afunction of wavelength for each sample analyzed.

In the absence of NaCl or presence of 10 mM or 25 mM NaCl, no bindingbetween the dsDNA targets (SEQ ID NO:1 or SEQ ID NO:2) and theantiparallel ssDNA-F Probe No. 7 was detected, after all incubationperiods (data not shown).

After a 1 hour incubation in the presence of 50 mM NaCl, dsDNA:ssDNA-Fcomplexes consisting of perfectly complementary sequences (SEQ IDNO:1+°Probe No. 7) formed readily, resulting in a 49% decrease influorescent emission intensity compared to that emitted by the controlProbe No. 7, which was similarly incubated in the reaction mixture (FIG.13A). By contrast, incompletely complementary dsDNA:ssDNA-F complexescontaining a 1 bp G-T mismatch (SEQ ID NO:2+Probe No. 7) yielded a 11%decrease in fluorescent emission intensity compared to that exhibited bythe Probe No. 7 control sample.

The presence of 75 mM, 100 mM and 125 mM NaCl in the reaction mixturealso resulted in fluorescent emission quenching consistent withsignificant amounts of complex formation between the perfectly matchedSEQ ID NO:1 target and antiparallel Probe No. 7, and significantly lessquenching when the 1 bp G-T mismatched SEQ ID NO:2 target and Probe No.7 were present, producing similar fluorescent intensities to thatobserved in the presence of 50 mM NaCl (data not shown).

DsDNA:ssDNA complexes were also formed when the parallel ssDNA Probe No.6 was reacted with the 50-mer wild-type dsDNA target (SEQ ID NO:5) andmutant dsDNA target (SEQ ID NO:7) in the presence of 50 mM, 75 mM, 100mM or 150 mM NaCl. Optimum results were obtained in the presence of 100mM NaCl. After a 75 minute incubation at room temperature in a reactionmixture containing 100 mM NaCl, the fluorescent emission intensities fora perfectly matched DNA complex (SEQ ID NO:5+Probe No. 6) and a 1 bpmismatched DNA complex (SEQ ID NO:7+Probe No. 6) were 53% and 9% lower,respectively, than that obtained by the control parallel ssDNA Probe No.6 reacted under the same conditions (FIG. 13B). 50 mM NaCl promotedmaximum discrimination between perfectly matched and mismatchedcomplexes in an incubation period of 45 minutes (data not shown). Ingeneral, complexes containing either antiparallel or parallel ssDNAprobes seemed to form with similar efficiencies at similar NaClconcentrations and incubation periods.

Use of monovalent cations, which are known DNA condensing agents,facilitated DNA complex formation between non-denatured dsDNA targetsand fluorescently-labeled antiparallel or parallel ssDNA probes, toallow reliable differentiation between complexes containing perfectlycomplementary sequences and those containing 1 bp mismatches.

Example 14

DsDNA:ssDNA complexes facilitated by YOYO-1 readily form at roomtemperature within 5 minutes of incubation and generate fluorescentemissions at the same level of intensity for hours. Complexes containingbase pair mismatches similarly emit fluorescent signals which persist,indicating the same level of complex formation over time. To examine therate of formation, stability and rate of disassociation of dsDNA:ssDNAcomplexes formed in the presence of condensing agents such as cations,time course experiments were performed.

Each hybridization reaction mixture (40 μl) contained the following: 0.4pmoles of non-denatured target dsDNA, 4 pmoles of 5′-fluorescein labeledssDNA probe, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 10 mM MnCl₂. Thereaction mixtures were incubated at room temperature (21° C.) forvarious periods ranging from 1 minute to 2 hours. Following incubation,samples were placed into a quartz cuvette, irradiated once with an argonion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. Further fluorescent measurements were taken of thesame samples after subsequent multiple laser irradiation, at theindicated times (FIG. 14). The intensity of fluorescence was plotted asa function of time for each sample analyzed.

The fluorescence emitted by control samples comprising 4 pmoles of ProbeNo. 5 plus 10 mM MgCl₂ and 10 mM MnCl₂, in the absence of target dsDNA,dramatically decreased 3-fold within just 5 minutes of incubation (datanot shown), and then steadily declined at a much slower rate within thenext few hours (FIG. 14A). This effect we refer to as “Cationic Quench”.This inhibition of fluorescence, associated with increased incubationperiods of ssDNA-F probes with specific cations, occurred routinely inthe presence of divalent cations, but not in the presence of monovalentcations (data not shown). This observation makes evident the importanceof incubating the control sample in an experiment under exactly the sameconditions that the test samples of an experiment are reacted. Multiplelasing of each ssDNA-F control sample after varying periods ofincubation inhibited further quenching of the fluorophore, resulting ina steady level of fluorescence thereafter (FIG. 14A). This result wasentirely unanticipated.

When the antiparallel ssDNA-F Probe No. 5 was incubated with the 50-merwild-type dsDNA target (SEQ ID NO:5) in the presence of 10 mM MgCl₂ and10 mM MnCl₂, dsDNA:ssDNA-F complex formation was evident after 15minutes of incubation resulting in a decrease in fluorescence, which was6% greater than the progressive cationic quench of the control Probe No.5 (compare FIGS. 14A and 14B). Complex formation was greatly indicatedafter 30 and 60 minutes of incubation of SEQ ID NO:5 with Probe No. 5 inthe presence of 10 mM MgCl₂ and 10 mM MnCl₂, generating a 76% and 61%decrease in fluorescence, respectively, compared to that achieved by thecationically quenched Probe No. 5 alone (FIG. 14B). After 90 and 120minutes of incubation in the presence of 10 mM MgCl₂ and 10 mM MnCl₂, nocomplex formation was being signaled (FIG. 14B) The level of fluorescentemission seen at 90 and 120 minutes was wholly attributable to thecationic quench effect (compare FIGS. 14A and 14B).

By contrast, dsDNA:ssDNA-F complexes that contained a 1 bp T-G mismatch(SEQ ID NO:7+Probe No. 5) formed at a slower rate and were much lessstable once formed in the presence of 10 mM MgCl₂ and 10 mM MnCl₂. The 1bp T-G mismatched complex was first observed after 30 minutes ofincubation, and appeared to have been eliminated after 60 minutes ofincubation (FIG. 14C). Once again, the probe was antiparallel to thecomplementary strand in the duplex (FIG. 14C).

Multiple laser irradiation of perfectly complementary dsDNA:ssDNAcomplexes (SEQ ID NO:5+Probe No. 5) formed after 30 minutes or 60minutes of incubation in the presence of 10 mM MgCl₂ and 10 mM MnCl₂resulted in fluorescent emissions consistent with the destruction ofthese complexes at a rate characteristic for DNA complexes containing anantiparallel ssDNA probe (FIG. 14B). When a subsequent measurement wasmade at 45 minutes after lasing of the perfectly complementary complexat 30 minutes, the emission intensity level was 1869, testimony to therapidity with which the complex was destroyed (data not shown). Thelevel of fluorescent emission, after multiple lasing, returned to thecationically quenched values observed by the uncomplexed Probe No. 5alone control (compare FIGS. 14A and 14B). The only exception was theperfectly matched complexes formed after 15 minutes of incubation andrepeatedly irradiated thereafter (FIG. 14B). In this case thefluorescent emission was not consistent with the destruction of thecomplexes (FIG. 14B), even though further cationic quench of Probe No.5, when multiply irradiated after a 15 minute incubation, was totallyinhibited (FIG. 14A). DsDNA:ssDNA complexes containing a 1 bp T-Gmismatch (SEQ ID NO:7+Probe No. 5) were similarly apparently destroyedby multiple lasing (FIG. 14C).

An experiment was performed to determine the basis for the effect ofmultiple lasing on the complexes. It was found that when fresh cationswere added to the reaction mixture which had been lased twice, theinhibition of cationic quench in fluorescence emitted by the ssDNA-Fprobe could not be reversed and further cationic quench did not occurupon further incubation, strongly suggesting that the ssDNA-F probe wasinactivated by multiple irradiation, by a yet unknown mechanism (datanot shown). Similarly, when fresh ssDNA-F probes were added to thereaction mixture which had been lased twice, after normalizing for theincreased fluorescent emission of the fresh probe, no subsequentprogressive cationic quenching was observed upon further incubation ofthe reaction mixture, strongly suggesting that the lased cations weresomehow disabled (data not shown).

In the foregoing examples and description, we have elucidated thatheteropolymeric nucleic acid strands can specifically bind on the basisof homologous base pairing. Such binding can occur between parallel orantiparallel strands.

We have also elucidated that nucleic acid bases bound in a Watson-Crickcomplementary duplex are not quiescent as regards the bases of proximalnucleic acid strands and that such bases can be interacted with on thebasis of Watson-Crick complementary base pairing or homologous basepairings, depending on the binding potential of the proximal sequence ofbases determined by either of the possible binding motifs. This is truewhether the bases in the duplex are interacting with bases in a thirdstrand to form a specifically bound triplex structure or whether thebases of the duplex are specifically interacting with proximal baseswhich are themselves coupled into a Watson-Crick complementary duplex.Accordingly the invention comprises the discovery that Watson-Crickcoupled bases remain reactive as specific bases to interact and bind toproximal bases on other strands and do so with great specificity andalacrity. While all of this is remarkable, it is considered especiallyremarkable that A:T and G:C pairings are detected as mismatches inbinding reactions wherein the homologous binding motif is dominant andbeing enforced on all base pairs by a strand-wide imperative. It islikewise remarkable that homologous quadruplex binding is more specificthan is Watson-Crick complementary quadruplex binding. Of necessityquadruplex binding occurs between the major groove side of aduplex-coupled base or base pair and the minor groove side of aduplex-coupled base or base pair. Heretofore, while the potential offurther binding by a base already complexed in a duplex was unknown, ithad been postulated that third strand recognition of bases in a duplexoccurred solely in the major groove of the duplex. This we show is notthe case. We have also demonstrated that putative backbone repulsion isno barrier to duplex:duplex interaction.

Our invention relates to readily achieved binding reactions which aretypically achieved with short incubation periods at room temperature andwhich do not depend on molar excess of a reagent to drive a reaction.Accordingly the invention is shown to be not only readily achieved, butobviously biologically relevant.

Finally we have shown that partial Watson-Crick complementaryconjugation with free bases can contribute to increased duplex bindingand increased specificity.

The invention constitutes a substantial addition to the knowledge ofbase binding and as such will be central to the elucidation of manybiological functions whose mechanisms are currently mysterious, such asgene silencing.

It is most remarkable to detect specific homologous recognition andbinding by bases previously and stably coupled into Watson-Crickcomplementary duplex.

We believe that what we have elucidated will require the abandonment ofmany “canonical” ideas and the reopening of the question of nucleic acidbinding capability.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A complex comprising: a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues containing purines and pyrimidines; and a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues containing purines and pyrimidines, wherein: (a) at least one of said probe and said target is purified or synthetic; (b) bonding of said heteropolymeric probe sequence to said heteropolymeric target sequence consists of: (i) Watson-Crick bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence; or (ii) homologous bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence; and (c) all bases of said probe and said target are selected from the group consisting of A, T, G, C and U, provided that when said complex is a duplex and said heteropolymeric probe sequence is antiparallel to said heteropolymeric target sequence, said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction, and provided that when said complex is a triplex, said complex is free of recombination proteins.
 2. A complex comprising: a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of said probe and said target is purified or synthetic; (b) said complex is a duplex; (c) said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality; and (d) said heteropolymeric probe sequence is bound to said heteropolymeric target sequence by Watson-Crick complementary base interaction.
 3. The complex of claim 1, wherein one of said probe and said target is single stranded, the other of said probe and said target is double-stranded, and said complex is a triplex.
 4. The complex of claim 3, wherein said complex is free of PNA.
 5. The complex of claim 3, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction.
 6. The complex of claim 3, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction.
 7. The complex of claim 3, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality; (b) said target includes a first strand containing said heteropolymeric target sequence and a second strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (c) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction and said heteropolymeric probe sequence is bonded to said second heteropolymeric target sequence by Watson-Crick complementary base interaction.
 8. The complex of claim 3, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have antiparallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction.
 9. The complex of claim 3, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have antiparallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction.
 10. The complex of claim 3, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have antiparallel directionality; (b) said target includes a first strand containing said heteropolymeric target sequence and a second strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (c) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction and said heteropolymeric probe sequence is bonded to said second heteropolymeric target sequence by Watson-Crick complementary base interaction.
 11. A complex comprising: a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of said probe and said target is purified or synthetic; (b) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction; and (c) said probe and said target are double stranded and said complex is a quadruplex.
 12. The complex of claim 11, wherein said complex is free of PNA.
 13. The complex of claim 11, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction.
 14. The complex of claim 11, wherein said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality, and said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction.
 15. The complex of claim 11, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality; (b) said probe includes a first probe strand containing said heteropolymeric probe sequence and a second probe strand containing a second heteropolymeric probe sequence complementary and antiparallel to said heteropolymeric probe sequence; (c) said target includes a first target strand containing said heteropolymeric target sequence and a second target strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (d) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction and said heteropolymeric probe sequence is optionally bonded to said second heteropolymeric target sequence by Watson-Crick complementary base interaction.
 16. The complex of claim 11, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have parallel directionality; (b) said probe includes a first probe strand containing said heteropolymeric probe sequence and a second probe strand containing a second heteropolymeric probe sequence complementary and antiparallel to said heteropolymeric probe sequence; (c) said target includes a first target strand containing said heteropolymeric target sequence and a second target strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (d) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction and said heteropolymeric probe sequence is optionally bonded to said second heteropolymeric target sequence by homologous base interaction.
 17. The complex of claim 11, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have antiparallel directionality; (b) said probe includes a first probe strand containing said heteropolymeric probe sequence and a second probe strand containing a second heteropolymeric probe sequence complementary and antiparallel to said heteropolymeric probe sequence; (c) said target includes a first target strand containing said heteropolymeric target sequence and a second target strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (d) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction and said heteropolymeric probe sequence is optionally bonded to said second heteropolymeric target sequence by Watson-Crick complementary base interaction.
 18. The complex of claim 11, wherein: (a) said heteropolymeric probe sequence and said heteropolymeric target sequence have antiparallel directionality; (b) said probe includes a first probe strand containing said heteropolymeric probe sequence and a second probe strand containing a second heteropolymeric probe sequence complementary and antiparallel to said heteropolymeric probe sequence; (c) said target includes a first target strand containing said heteropolymeric target sequence and a second target strand containing a second heteropolymeric target sequence complementary and antiparallel to said heteropolymeric target sequence; and (d) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by Watson-Crick complementary base interaction and said heteropolymeric probe sequence is optionally bonded to said second heteropolymeric target sequence by homologous base interaction.
 19. The complex of claim 1, wherein said probe occupies a minor groove of said target.
 20. The complex of claim 1, wherein said probe occupies a major groove of said target.
 21. The complex of claim 3, wherein bases of said probe interact with bases of both strands of said target simultaneously.
 22. The complex of claim 11, wherein bases of said probe interact with bases of both strands of said target simultaneously.
 23. The complex of claim 1, wherein a backbone of the probe comprises deoxyribose phosphate.
 24. The complex of claim 1, wherein a backbone of the probe is uncharged, partially negatively charged, or positively charged.
 25. The complex of claim 1, wherein at least one base in said probe is a synthetic analogue of a naturally occurring base having specific binding affinities based on Watson-Crick complementarity or homologous binding.
 26. A complex comprising: a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues: and a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues. wherein: (a) at least one of said probe and said target is purified or synthetic; (b) said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by base interaction between bases of said probe and bases of said target, said base interaction being Watson-Crick complementary base interaction or homologous base interaction; (c) the probe further contains a loop and a stem adjacent to the loop; (d) the heteropolymeric probe sequence is located partially or entirely within the stem; and (e) each of the loop and the stem comprises nucleic acids or nucleic acid analogues, provided that when said complex is a duplex and said heteropolymeric probe sequence is antiparallel to said heteropolymeric target sequence, said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction, and provided that when said complex is a triplex, said complex is free of recombination proteins.
 27. A method for making a complex of claim 1, said method comprising: providing a sample comprising the target containing the heteropolymeric target sequence of nucleic acids or nucleic acid analogues; providing the probe, wherein: (a) the probe further contains a loop and a stem adjacent to the loop; (b) the heteropolymeric probe sequence is located within at least one of the loop and the stem; and (c) each of the loop and the stem comprises nucleic acids or nucleic acid analogues; and forming a reaction mixture comprising the probe and the target under conditions that the probe and the target hybridize to form the complex.
 28. The method of claim 27, further comprising detecting a signal correlated with a binding affinity between the probe and the target such that the target is detected.
 29. The method of claim 28, wherein a match or a mismatch between bases of the heteropolymeric probe sequence and bases of the heteropolymeric target sequence is detected.
 30. The method of claim 27, wherein the probe and the target hybridize to form a duplex.
 31. The method of claim 27, wherein one of the heteropolymeric probe sequence and the heteropolymeric target sequence is single stranded, the other of the heteropolymeric probe sequence and the heteropolymeric target sequence is double-stranded, and the probe and the target hybridize to form a triplex.
 32. The method of claim 27, wherein the heteropolymeric probe sequence and the heteropolymeric target sequence are double stranded and the probe and the target hybridize to form a quadniplex.
 33. The method of claim 27, wherein: (a) homologous binding conditions are provided and an incompletely homologous probe does not bind to the target or binds to the target with reduced efficiency; or (b) Watson-Crick complementary binding conditions are provided and an incompletely complementary probe does not bind to the target or binds to the target with reduced efficiency.
 34. The method of claim 27, wherein the heteropolymeric probe sequence is located in the stem.
 35. The method of claim 27, wherein the heteropolymeric probe sequence is located in the loop.
 36. The method of claim 35, wherein: (a) the stem comprises at least two strands of nucleic acids or nucleic acid analogues, (b) the at least two strands are adapted to be bound to each other in the absence of the heteropolymeric target sequence binding to the heteropolymeric probe sequence, and (c) the at least two strands are adapted to separate from each other when the heteropolymeric target sequence binds to the heteropolymeric probe sequence.
 37. The method of claim 36, wherein: (a) one of the at least two strands comprises a donor; (b) an opposing one of the at least two strands comprises an acceptor; and (c) the method further comprises detecting a change in a signal when the heteropolymeric target sequence binds to the heteropolymeric probe sequence and the at least two strands separate from each other.
 38. The method of claim 37, wherein: (a) the donor emits the signal and the acceptor quenches the signal; or (b) the donor transfers energy to the acceptor and the acceptor emits the signal.
 39. The method of claim 38, wherein the energy transferred is fluorescent resonance energy.
 40. The method of claim 38, wherein the signal is chemiluminescent.
 41. The complex of claim 1, wherein: (a) the probe is a heteropolymeric multistranded parallel complex; (b) bases of the probe are homologously bound in a first plane; (c) the target is a heteropolyineric multistranded parallel complex; and (d) bases of the target are homologously bound in a second plane.
 42. The complex of claim 1, wherein: (a) the probe is a heteropolymeric multistranded parallel complex; (b) bases of the probe are bound pursuant to a Watson-Crick motif in a first plane; (c) the target is a heteropolymeric multistranded parallel complex; and (d) bases of the target are bound pursuant to a Watson-Crick motif in a second plane.
 43. An isolated or synthetic probe comprising: a loop containing nucleic acids or nucleic acid analogues; a stem adjacent to the loop, and containing at least two strands of nucleic acids or nucleic acid analogues; and a heteropolymeric probe sequence located entirely or partially within the loop, and containing a double or triple-stranded sequence of nucleic acids or nucleic acid analogues, wherein the at least two strands of the stem are adapted to be bound to each other in the absence of the heteropolymeric target sequence binding to the heteropolymeric probe sequence, and the at least two strands are adapted to separate from each other and open the loop when a heteropolymeric target sequence binds to the heteropolymeric probe sequence.
 44. The complex of claim 11, wherein said complex is free of Hoogsteen binding and free of G-G quartets.
 45. A complex comprising: a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of said probe and said target is purified or synthetic; (b) bonding of said heteropolymeric probe sequence to said heteropolymeric target sequence consists of: (i) Watson-Crick bonds comprising at least two members selected from the group consisting of an A-T bond, an A-U bond, and a G-C bond, or (ii) homologous bonds comprising at least four members selected from the group consisting of an A-A bond, a T-T bond, a G-G bond, a C-C bond and a U-U bond; and (c) all bases of said probe and said target are selected from the group consisting of A, T, G, C and U, provided that when said complex is a duplex and said heteropolymeric probe sequence is antiparallel to said heteropolymeric target sequence, said heteropolymeric probe sequence is bonded to said heteropolymeric target sequence by homologous base interaction, and provided that when said complex is a triplex, said complex is free of recombination proteins.
 46. The complex of claim 11, wherein said heteropolymeric probe sequence comprises purines and pyrimidines, said heteropolymeric target sequence comprises purines and pyrimidines, all bases of said probe and said target are selected from the group consisting of A, T, G, C and U, and bonding of said heteropolymeric probe sequence to said heteropolymeric target sequence consists of: (i) Watson-Crick bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence; or (ii) homologous bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence.
 47. The complex of claim 26, wherein said heteropolymeric probe sequence comprises purines and pyrimidines, said heteropolymeric target sequence comprises purines and pyrimidines, all bases of said probe and said target are selected from the group consisting of A, T, G, C and U, and bonding of said heteropolymeric probe sequence to said heteropolymeric target sequence consists of: (i) Watson-Crick bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence; or (ii) homologous bonds between each pyrimidine of the heteropolymeric probe sequence and each opposing pyrimidine of the heteropolymeric target sequence, and between each purine of the heteropolymeric probe sequence and each opposing purine of the heteropolymeric target sequence. 